Veterinary Microbiology 114 (2006) 173–186 www.elsevier.



The role of probiotics in aquaculture
´ ´ Jose Luis Balcazar a,*, Ignacio de Blas a, Imanol Ruiz-Zarzuela a, ´ ´ David Cunningham b, Daniel Vendrell a, Jose Luis Muzquiz a

Laboratory of Fish Pathology, University of Zaragoza, c/. Miguel Servet 177, 50013 Zaragoza, Spain b Environmental Science and Occupational Safety Lab, Massachusetts Bay Community College, Wellesley, MA, USA Received 26 April 2005; received in revised form 7 November 2005; accepted 17 January 2006

Abstract The increase of productivity in aquaculture has been accompanied by ecological impacts including emergence of a large variety of pathogens and bacterial resistance. These impacts are in part due to the indiscriminate use of chemotherapeutic agents as a result of management practices in production cycles. This review provides a summary of the use of probiotics for prevention of bacterial diseases in aquaculture, with a critical evaluation of results obtained to date. # 2006 Elsevier B.V. All rights reserved.
Keywords: Probiotics; Bacteria; Disease control; Aquaculture

Contents 1. 2. 3. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . What is a probiotic?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of action. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Competitive exclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Source of nutrients and enzymatic contribution to digestion 3.3. Influence on water quality . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Enhancement of the immune response . . . . . . . . . . . . . . . 3.5. Antiviral effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Colonization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Selection of probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Consideration of regulations on probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174 175 175 175 176 176 176 177 177 177 178

4. 5.

* Corresponding author. Tel.: +34 976761569; fax: +34 976761612. ´ E-mail address: (J.L. Balcazar). 0378-1135/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2006.01.009

. . . . . . . . . . 2003). . . 1992). . Crustaceans . . .. . . . . . . . . . Although the original epidemic strain of Vibrio cholerae 01 was susceptible to the 12 antimicrobial agents tested. . . . . . . 1600 t of antibiotics. . . . . . . . . . . . . . . . . . . . . . . which control pathogens through a variety of mechanisms. . . transduction via viruses and even direct transformation from DNA absorbed to the particles in the water or on the sediment surfaces could all be likely mechanisms for genetic exchange (Moriarty. . . . Rinkinen et al. . . . . . . . . . ` 2002. . . . Irianto and Austin. . . . . . . . given extensive documentation of the evolution of antimicrobial resistance among pathogenic bacteria. . 6. . . . . is almost identical by molecular sequence to the florofenicol resistance gene first described in Photobacterium damsela. . . . In addition. . . . . . the utility of antimicrobial agents as a preventive measure has been questioned. 2003). . . . . . . . . . . . . . . . . . . .. . . they have begun to be applied in aquaculture (Gatesoupe. . . . . were similarly used for growth promotion purposes in 1997 (SCAN. . . .1. . References . . . . . . . . . . . . . . . . 12. 2000. . . . For example. . . . . . . . . . . . . . . in coastal Ecuador it became multidrug resistant by the transference of resistance genes of non-cholera vibrios that are pathogenic to the shrimp (Weber et al. . . .L. . . .174 ´ J. In the European Union and Switzerland.. .000 t of antibiotics produced each year for medical and agricultural purposes. . . . 2003) and recently. . . Several bacterial pathogens can develop plasmid-mediated resistance. 179 179 181 182 183 183 183 183 1. . . . Prevention and control of diseases have led during recent decades to a substantial increase in the use of veterinary medicines. . . . . . . . . . . . . . . . . . . . . . . . 6. . . . . . . . . . . . . . . Plasmids carrying genes for resistance to antibiotics have been found in marine Vibrio species and they could be laterally exchanged. The use of probiotics or beneficial bacteria. . . . 1997 reviews. . . . . Fish juveniles and adults . . . representing about 30% of the total use of antibiotics in farm animals. 1997). . . . . . . . . . . . 6. . . . . . mechanisms of action and criteria for selection of probiotics. . . . . . . . . . . . . transference of multidrug resistance occurred in Ecuador during the cholera epidemic (1991–1994) in Latin America and this began among persons who were working on shrimp farms. . . . . . . . . . . is increasingly viewed as an alternative to antibiotic treatment. Mollusks . . . . . . . . . . . . . . . 2000. . . . . . Resistance mechanisms can arise one of two ways: chromosomal mutation or acquisition of plasmids. Chromosomal mutations cannot be transferred to other bacteria but plasmids can transfer resistance rapidly (Lewin. . . . tonnes of antibiotics have been distributed in the biosphere during an antibiotic era of only about 60 years duration. . The use of probiotics in human and animal nutrition is well documented (see Fuller. . . . . . . . . . . . Introduction Aquaculture has become an important economic activity in many countries. . . .. . . . 7. . . and to summarize their applications in aquaculture. . . . where aquatic animals are exposed to stressful conditions. . . other evidence of the transmission of resistance between aquaculture ecosystems and human has been demonstrate. . . . . . . . . . . . . Acknowledgements . . . At the high population densities of bacteria found in aquaculture ponds. . . . .4. . . . . bacterium found in fish (Angulo. . . 2000). . . . . . . . . . . . . . . . . Fish eggs and larvae . In the United States. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . when a novel florofenicol resistance gene floR. . . . . The purposes of this review are to describe the principles. . . In large-scale production facilities. in Salmonella typhimurium DT104. . . . . 6. . . . . 2003). These amounts of antibiotics have exerted a very strong selection pressure towards resistance among bacteria. . . . .3. . . . . . Globally. . mainly by a horizontal and promiscuous flow of resistance genes (SCAN. . . . . . . . . which also confers resistance to chloramphenicol. . . . .2. . 1994). . . 2003). . . . . . Balcazar et al. . . 18. . . . . . . . Verschuere et al. . . . . . . . . . which have adapted to this situation. . . . . . . . . . . . . . . . However. . transfer via plasmids. Conclusions. . . . . . . 1992. . . . . . .600 t are used for the nontherapeutic treatments of livestock in order to promote growth (SCAN. 1999. . problems related to diseases and deterioration of environmental conditions often occur and result in serious economic losses. . . . . . . . . . . . . . Gomez-Gil et al. / Veterinary Microbiology 114 (2006) 173–186 6. .. Probiotic strains studied in aquaculture. . . . Bachere. . . . . . Mulder et al. . . . . . . . .

Balcazar. Moriarty. Balcazar. Several studies on probiotics have been published during the last decade. Prieur et al. representatives of the family Enterobacteriaceae. stated that probiotics are live microorganisms. Balcazar et al.1. ´ Gullian and Rodrıguez. Subsequently. Lilley and Stillwell (1965) described them as substances secreted by one microorganism. ´ ´ 2002. 2001). Mechanisms of action Enhancement of colonization resistance and/or direct inhibitory effects against pathogens are important factors where probiotics have reduced .. However.. Scholz et al. and obligate anaerobic bacteria of the genera Bacteroides. microbial interactions play a major role in the equilibrium between competing beneficial and potentially pathogenic microorganisms. Lactobacillus in swine. 1998). the indigenous microbiota of freshwater fish species tends to be dominated by members of the genera Aeromonas. Nevertheless. 1994). In contrast to saltwater fish.. nutritional and environment factors. Fusubacterium. 1995. An expert with the Joint Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO). (v) antiviral effects (Kamei et al.. The gut microbiota of aquatic animals is probably constituted by the indigenous microbiota jointly with artificially high levels of allochtonous microorganisms so maintained by their constant ingestion from the surrounding water (Hansen and Olafsen. which stimulated the growth of another. 1995. Probiotic strains have been shown to inhibit pathogenic bacteria both in vitro and in vivo through several different mechanisms. Rosenfeld and Zobell (1947) described a study of antibiotic-producing marine microorganisms. 1998). some possible benefits linked to the administering of probiotics have already been suggested as: (i) competitive exclusion of pathogenic bacteria (Garriques and Arevalo. Moriarty. 1995.. 2002. Vine et al. 3. 1997). therefore.. 2003. Gram-negative facultative anaerobic bacteria such as Vibrio and Pseudomonas constitute the predominant indigenous microbiota of a variety of species of marine fish (Onarheim et al. 1990. What is a probiotic? The term probiotics is generally used to denote bacteria that promote the health of other organisms. and 3. However. microbial manipulation constitutes a viable tool to reduce or eliminate the incidence ´ of opportunist pathogens (Balcazar. Enterococcus in carnivore). probiotic strains have been isolated from indigenous and exogenous microbiota of aquatic animals. which are prevalent in the mammal or bird gut (Bifidobacterium in human. 2004. 2004a). 1990). 1995). and others are still being investigated as: (iv) enhancement of the immune response against pathogenic microorganisms (Andlid et al. therefore. / Veterinary Microbiology 114 (2006) 173–186 175 2. ´ 1997.´ J. Balcazar et al. rodent and bird.. (iii) direct uptake of dissolved organic material mediated by the bacteria (Garriques and Arevalo.L. Direkbusarakom et al. Lactic acid producing bacteria.. 1988.. Generally. 2000. are generally sub-dominant in fishes and represented essentially by the genus Carnobacterium (Ringø and Vadstein.. (ii) source of nutrients and enzymatic contribution to digestion (Sakata. confer a health benefit for the host (FAO/WHO. 2004). Irianto and Austin. 1999. microorganisms present in the immediate environment of aquatic species have a much larger influence on the health status than with terrestrial animals or humans.. 2002). 2003. 1989). 1989. 2000. the composition of microbial communities can be altered by husbandry practices and environmental conditions that stimulate the proliferation of selected bacterial species. It is well known that the microbiota in the gastrointestinal tract of aquatic animals can be modified. for example by ingestion of other microorganisms. 1990. It is important to note that the population of endogenous microbiota may depend on genetic. ´ Balcazar et al. Garriques and Arevalo. Gomez-Gil et al. Girones et al. the incidence and duration of diseases. and Eubacterium (Sakata. has been attributed to Gaixa (1889). However. which when consumed in adequate amounts. the methodological and ethical limitations of animal studies make it difficult to understand the mechanisms of action of probiotics. and only partial explanations are available. Competitive exclusion Bacterial antagonism is a common phenomenon in nature. Rengpipat et al. Plesiomonas.. The first report of the existence in seawater of bacteria with an inhibitory effect against a Vibrio sp.

. / Veterinary Microbiology 114 (2006) 173–186 since then the research has started to develop biological control agents. Enhancement of the immune response The non-specific immune system can be stimulated by probiotics. 2000). Pseudomonas I2. In addition. Nogami et al. Influence on water quality Improved water quality has especially been associated with Bacillus sp.. stimulated the respiratory burst in rainbow trout (Oncorhynchus mykiss)..176 ´ J. It has been reported that use of Bacillus sp.. such as proteases. Recently a marine bacterial strain. may contribute to nutritional processes in Arctic charr (Salvelinus alpinus L. Subsequently. where a complement of enzymes for digestion and synthesize compounds that are assimilated by the animal (Wang et al. Thalassobacter utilis. 1990).. especially by supplying fatty acids and vitamins (Sakata.4. Brevibacterium sp. Microbiota may serve as a supplementary source of food and microbial activity in the tract digestive may be a source of vitamins or essential amino acids (Dall and Moriarty. This strain increased the survival of larvae of the crab. Portunus trituberculatus. 1992. (2000) mentioned that the use of Bacillus sp. Microbacterium sp. and resistant to proteolytic enzymes (Chythanya et al.. as well as providing necessary growth factors (Prieur et al. high levels of gram-positive bacteria can be minimize the buildup of dissolved and particulate organic carbon. 1995). suppressed the growth of the fish pathogen V. 3. 1995). In aquaculture. improved water quality. Rengpipat et al. 1983). (strain S11) provided disease protection by activating both cellular and humoral immune defenses in tiger ´ shrimp (P. it has been reported that Bacteroides and Clostridium sp. Source of nutrients and enzymatic contribution to digestion Some researches have suggested that microorganisms have a beneficial effect in the digestive processes of aquatic animals. Balcazar et al. In addition. (2003) showed that administration of a lactic acid bacterium Lactobacillus rhamnosus (strain ATCC 53103) at a level of $105 cfu gÀ1 feed.. Some microorganisms such as Agrobacterium sp. some bacteria may participate in the digestion processes of bivalves by producing extracellular enzymes. soluble in chloroform. 3.) (Ringø et al. heat stable. This antibacterial compound was shown to be of low molecular weight..2. 1995). in the water used to rear the larvae (Nogami and Maeda. Balcazar (2003) demonstrated that the administration of a mixture of bacterial strains (Bacillus and Vibrios sp.L. The use of Vibrio alginolyticus strains as a probiotics has been recommended to increase survival and growth of white shrimp (Litopenaeus vannamei) post-larvae in Ecuadorian hatcheries. In fish. The rationale is that grampositive bacteria are better converters of organic matter back to CO2 that gram-negative bacteria.. Similar observations have been reported for the microbial flora of adult penaeid shrimp (Penaeus chinensis). This protection was due to a stimulation of the immune system. 1990). have contributed to the host’s nutrition. was isolated from estuarine environmental samples that produced inhibitory compounds against shrimp pathogenic vibrios. 2002).. Pseudomonas sp.3. by increasing the phagocytic activity of leucocytes (Sakai et al. has shown inhibitory effects against Vibrio anguillarum. . by increasing phagocytosis and antibacterial activity. It has been demonstrated that oral administration of Clostridium butyricum bacteria to rainbow trout enhanced the resistance of fish to vibriosis. Competitive exclusion of potential pathogenic bacteria effectively reduces or eliminates the need for antibiotic prophylaxis in intensive larviculture systems (Garriques and Arevalo. 3. survival and growth rates and increased the health status of juvenile Penaeus monodon and reduced the pathogenic vibrios (Dalmin et al. 1997). 2001).. anguillarum (Olsson et al. Nikoskelainen et al. it has been reported that bacterial strains associated with intestinal and skin mucus of adult marine turbot (Scophthalmus maximus) and dab (Limanda limanda). monodon).. and Staphylococcus sp. and also reduced the amount of Vibrio sp. During the production cycle.) positively influenced the growth and survival of juveniles of white shrimp and presented a protective effect against the pathogens Vibrio harveyi and white spot syndrome virus. lipases. 1992).

4. 2002.. laboratory tests indicate that the inactivation of viruses can occur by chemical and biological substances.. Direkbusarakom et al. bacterial growth may be inhibited by any antimicrobial compound produced by the host. 2000. Girones et al. NICA 1030 and NICA 1031 from a black tiger shrimp hatchery. 1988. For example. 5. and before the definitive installation of a very competitive indigenous microbiota. (iii) random amplification of polymorphic DNA ‘‘RAPD’’. 1991). 1988). bacteriocins.. normally colonize the hepatopancreas of juvenile white shrimp. with high specificity for poliovirus. followed by association within the mucous gel or attachment to epithelial cells. proteases. 1999. 2002) medium. According to Conway (1996). showed antiviral capacity. lysozymes. Vine et al. and groups of coryneforms isolated from salmonid hatcheries... Although the exact mechanism by which these bacteria do this is not known. showed antiviral activity against infectious hematopoietic necrosis virus (IHNV) with more than 50% plaque reduction (Kamei et al. In addition. Selection of probiotics A common way to select probiotics is to perform in vitro antagonism tests. diacetyl. 1998). a microorganism is able to colonize the gastrointestinal tract when it can persist there for a long time. however. enzymes. and genetic resistance.L. Cunningham. in which pathogens are exposed to the candidate probiotics or their extra´ cellular products in a liquid (Sotomayor and Balcazar. (iv) amplified fragment length polymorphism ‘‘AFLP’’. (v) terminal restriction fragment length polymorphism ‘‘T-RFLP’’.... 2002. Gram et al. 2002). bacteria may enter through the mouth. It has been reported that strains of Pseudomonas sp.. Balcazar et al. redox potential levels.. by possessing a multiplication rate that is higher than its expulsion rate. formation of ammonia. Antiviral effects Some bacteria used as candidate probiotics have antiviral effects. at which point some of them are retained as part of a resident microflora. 2004). respectively. or immune modulation (Salminen et al. The same methods employed for bacterial detection are used to study colonization. The process of colonization is characterized by attraction of bacteria to the mucosal surface. For example. Vibrio sp. and are eliminated via the faeces. Chythanya et al.´ J. Colonization Colonization of the gastrointestinal tract of animals by probiotics is possible only after birth. These isolates displayed antiviral activities against IHNV and Oncorhynchus masou virus (OMV). . 2004a) or solid (Dopazo et al. and (vii) denaturing gradient gel electrophoresis of PCRamplified 16S rDNA.5. Adhesion and colonization of the mucosal surfaces are possible protective mechanisms against pathogens through competition for binding sites and nutrients (Westerdahl et al.. either with water or food particles. which been recently introduced in microbial ecology (Spanggaard et al. Aeromonas sp. (vi) immunohistochemical methods. / Veterinary Microbiology 114 (2006) 173–186 177 3. However. (ii) Microbe-related factors: effects of antagonistic microorganims.. 1988.. only the addition of high doses of probiotic provokes its artificial and temporary dominance. For example. molecular techniques. Sugita et al. (1989) reported that a marine bacterium. this normal microflora can artificially become dominated by Bacillus sp. Vibrios sp. tentatively classified in the genus Moraxella. 2003. 1992). such as extracts from marine algae and extracellular agents of bacteria. lactic acid bacteria are known to produce compounds such as bacteriocins that inhibit the growth of other microorganisms. (i) immunocolony blot ‘‘ICB’’ and (ii) enzyme-linked immunosorbent assay ‘‘ELISA’’.. (1998) isolated two strains of Vibrio spp. Gullian et al. After this installation. and alteration of pH values by the production of organic acids (Dopazo et al. the population of probiotic organisms in the gastrointestinal tract shows a sharp decrease within days after the intake had stopped (Fuller.. Temmerman et al. 2003). In mature animals. Chythanya et al. with percentages of plaque reduction between 62 and 99%. Factors known to influence the colonization of microorganisms can be grouped as follows: (i) hostrelated factors: body temperature. (up to 50% of the total) if it is added to ´ the water for 20 days (Gullian and Rodrıguez.. 2002. Others are destroyed by the digestive process or pass through the gut. and pass down the alimentary tract. Gram et al. hydrogen peroxide. Common techniques include immunoassays.

1999). (ii) acquisition of potential probiotics. After a phase out of the use of antibiotics as growth promoters in animals. 1995. (iii) evaluation of the ability of potential probiotics to out-compete pathogenic strains. (iv) addition to artificial diet (Rengpipat et al. 178/2002 of the European Parliament was published. and environmental user. the White Paper on Food Safety and the Regulation (EC) No. in vitro antagonism of Pseudomonas fluorescens (strain AH2) against Aeromonas salmonicida does not confer Atlantic salmon protection against furunculosis. Diagram for selection of probiotics as biocontrol agents in aquaculture. Gram et al. a manufacturer is required to submit a dossier containing data and studies demonstrating the efficacy and safety of the product for animals. Therefore. potential probiotics must exert its beneficial effects (e. safety (non-pathogenic) and ability of the strain to survive to the transit through the gastrointestinal tract of the host (e. animal health and welfare. To obtain authorization. it must be authorized in accordance with the provisions of the Directive.g. that is.. Finally. (iii) addition to culture water (Moriarty. and proteases). 2000) (see Fig.. from primary production to the safety of animal feed. 1998). For example. modified in the European Union. marketing and use of feed additives are currently regulated under Council Directive 70/524/EEC.. / Veterinary Microbiology 114 (2006) 173–186 (1999) suggested that in vitro activity in well-diffusion assays and broth cultures cannot be used to predict a possible in vivo effect. enhanced nutrition and increased immune response) in the host. (iv) assessment of the pathogenicity of the potential probiotics. resistance to bile salts. the basis on which the utilization of feed additives was consolidated has been Fig. In conclusion. The ability of microorganisms to colonize is often considered as one of the main selection criteria for potential probiotics. In addition. the efficient adherence to intestinal epithelial cells to reduce or prevent colonization of pathogens (Vine et al. The authorization. Spanggaard et al. consumers and the environment. the probiotic must be viable under normal storage conditions and technologically suitable for industrial processes (e. 1). (v) evaluation of the effect of the potential probiotics in the host. it is essential to know the origin (it is preferable to use strains isolated from the host).. in order to establish a policy of food safety for the European Union and the European Food Safety Authority (EFSA) was created (Regulation (EC) No. EFSA works on all stages of food production and supply. Probiotics can be provided to the host or added to its aquatic environment in several ways: (i) addition via live food (Gomez-Gil et al. 2004b). 5. Balcazar et al. vannamei) tanks with probiotic bacteria at a density of 105 cfu mlÀ1 prevented colonization by pathogenic bacteria during ´ larval culture (Peeters and Rodrıguez. efforts have been directed towards providing assurance of a high level of protection of human health. 1.178 ´ J. Consideration of regulations on probiotics In the last few years.. and consumer interests. ... 1999).g. lyophilized). low pH.L. In terms of the additives used in feed products. 2001). (ii) bathing (Austin et al. 2001).g. right through the supply of food to consumers.. 1998.1. 2000). Before a feed additive may be marketed or used. 178/2002). conferring protection against vibriosis (Gram et al. it has been reported that daily inoculations of larval white shrimp (L. (vi) economic cost/benefit analysis (Gomez-Gil et al. For example. but is an effective probiotic in rainbow trout. the methods to select probiotic bacteria for use in aquaculture include: (i) collection of background information.

The approval process to obtaining a licence proceeds as follows. other probiotics are commercialized on the market that has been notified. labelling and health statements made on conventional foods. alginolyticus). to the genus Bacillus. but that do not appear in the last authorized list of feed additives published by the Commission (Council Directive 70/ 524/EEC. named ‘‘foods for specified health use’’ (FOSHU). and how this substance helps the consumer. In addition. toyoi Bacillus licheniformis Bacillus subtilis Enterococcus faecium Lactobacillus casei Lactobacillus farciminis Lactobacillus plantarum Lactobacillus rhamnosus Pediococcus acidilactici Saccharomyces cerevisiae Streptococcus infantarius 179 Recently. the petitioner must present a complete explanation of how the substance has been authorized for use in the food supply.´ J. and an optimum level of the particular substance to be consumed. First. some microorganisms have been authorized for use as probiotics in feedingstuffs in the European Union. 1998). the petition must include copies of computer literature searches done by the petitioner. nutritional composition. potential adverse effects for any segments of the population. before a health claim petition can be prepared and submitted. 2004). These communities can also be influenced by inorganic and organic compounds. substantial scientific evidence is needed of the health benefits of the product. the amount of the substance that is present in foods must be obtained from representative samples using methods from the Association of Official Analytical Chemists (AOAC). safety. the summary must establish a significant scientific agreement among experts qualified on the health claim. In addition. 6. and other nutritional or health factors that could interact with substances or food components of interest. 1998). 2005) and proceed as follows. although other genera or species have also been mentioned (Aeromonas and Flavobacterium) (Tables 2 and 3). 6. if no AOAC method is available. and dietary supplements. and those appearing in the process of notification demanded by the regulation are detailed in Table 1. to the genus Vibrio (V. 1995. Obviously. food for special dietary use. For summary of scientific data. Shinohara. . For preliminary requirements. one or more models that represent label statements must include a brief statement of the relevant conclusions of the summary.1. safety. appropriated level of intake. and finally by a committee appointed by the Ministry of Health and Welfare. and other food science issues (Berner and O’Donnell. First. 1996. physicochemical properties. Japan possesses a programme for the regulation of functional foods. Fish eggs and larvae The spawning and early life stages of fish larvae may have profound implications for the dynamics of microbial communities.L. which can approve the application (Arai. by academic experts. / Veterinary Microbiology 114 (2006) 173–186 Table 1 List of microorganism authorized as probiotics in feedingstuffs under Council Directive 70/524/EEC Probiotics Bacillus cereus var. a good manufacturer compiles scientific data on the health effects. copies of all articles cited. any interested person may petition FDA to issue a regulation regarding a health claim. the Food and Drug Administration (FDA) regulates the safety. the petitioner must submit the assay method used and data establishing the validity. and all information on adverse consequences to any segment of the population. For analytical data. medicinal foods. or to the genus Pseudomonas. The general requirements for a petition are detailed in the US Code of Federal Regulations (CFR. For model health claims. the application is submitted to the Ministry of Health and Welfare. and test methods for the food or compound of interest. Berner and O’Donnell. Balcazar et al. The application is evaluated by the Japan Health Food and Nutrition Food Association. In the US. with the above information plus descriptive information. Then. Probiotic strains studied in aquaculture Most probiotics proposed as biological control agents in aquaculture belong to the lactic acid bacteria (Lactobacillus and Carnobacterium).

1989). / Veterinary Microbiology 114 (2006) 173–186 Table 2 Probiotics considered as biological control agents in aquaculture of fishes Probiotic strain Streptococcus lactis and Lactobacillus bulgaricus Lactobacillus sp. (1999) Robertson et al. A significant increase in the mean weight and survival rate of turbot larvae (S. rhamnosus JCM 1136 Roseobacter sp. (2004) Ghosh et al. (1995) Gildberg and Mikkelsen (1998) Queiroz and Boyd (1998) Turbot Oreochromis niloticus Rainbow trout (Oncorhynchus mykiss) Atlantic salmon Rainbow trout Rainbow trout Ringø and Vadstein (1998) Naik et al. Vibrio splendidus and a . fish larvae come into contact with their immediate environment that provides colonization by a wide variety of microorganism. Under normal conditions. (1999) Gram et al. Lactobacillus rhamnosus ATCC 53103 Aeromonas hydrophila.) Atlantic cod fry Channel catfish Method of application Enrichment of live food Enrichment of rotifers Bathing in bacterial suspension Addition to diet Addition to pond water Addition to culture water Addition to diet Addition to culture water Addition to diet Addition to diet Addition to diet Reference ´ Garcıa de la Banda et al. B. (1992) Gatesoupe (1994) Austin et al. The composition of microbiota is influenced by many factors including the availability of nutrients. and Carnobacterium sp. During the initial feeding period.. it is possible to manipulate the establishment of an artificial dominance of a determined group of bacteria in the fishassociated microbiota by adding a specific strain. It is therefore evident that health status will depend on management conditions to constitute the egg microbiota an important factor in the first establishment of an indigenous microbiota. Viable counts from water in the hatching units have been on the order of 103 mlÀ1 prior to hatching and 106 mlÀ1 2 days after hatching during early life of fish larvae in intensive rearing systems (Hansen and Olafsen. maximus) fed rotifers enriched in lactic acid bacteria and these strains provided a significant protection against a pathogenic Vibrio compared to control larvae (Gatesoupe. 1998). the addition of Carnobacterium divergens. subtilis.L. Vibrio fluvialis. anguillarum. Similarly.180 ´ J. Vibrio alginolyticus Carnobacterium divergens Bacillus megaterium. Carnobacterium sp. morhua) fry after a challenge with V. Tetraselmis copepod-fed larvae Intestines of Labeo rohita Anguilla anguilla Rainbow trout Turbot larvae L. polymyxa. (2004) Hjelm et al. 1994). animal physiology and immunological factors. showed a certain improvement in disease resistance in cod (G. rohita Addition to diet Addition to diet Addition to culture water Addition to diet Chang and Liu (2002) Panigrahi et al. one of the basic physiological functions of the resident microbiota is that it functions as a microbial barrier against microbial pathogens and as a complement to the establishment of digestive enzymes. (2000) Nikoskelainen et al. licheniformis Vibrio pelagius G-probiotic Pseudomonas fluorescens Carnobacterium sp. 34 strains of 400 marine bacteria exhibited in vitro antibacterial activity against V. (2004) Immediately after hatching process. Micrococcus luteus Enterococcus faecium SF68 L. (2001) Irianto and Austin (2002) Commercial product (Cernivet) Culture collection Turbot larvae. strain 27-4 Bacillus circulans Source ? Rotifers (Brachionus plicatilis) Commercial shrimp hatchery Intestines of Atlantic salmon Commercial product (Biostart) Turbot larvae Commercial product Iced freshwater fish (Lates niloticus) Intestines of Atlantic salmon Culture collection Digestive tract of rainbow trout Used on Turbot larvae (Scophthalmus maximus) Turbot larvae Atlantic salmon (Salmo salar L. Balcazar et al. A study in the rearing of larvae turbot. anguillarum (Gildberg and Mikkelsen. B. B.

S. salmonicida. Vibrio P62. it was reported that a commercially prepared bacterial mixture of Bacillus spp. and Yersinia ruckeri in fry and fingerling salmonids. (2004) Alavandi et al. (1999) Scholz et al. Cultures of Aeromonas hydrophila. alginolyticus C7b Pediococcus acidilactici Source Penaeus monodon Commercial product (DMS) Digestive tract of chicken Commercial product Used on P. applied at 5 Â 107 cells gÀ1 of feed (Robertson et al. Leuconostoc. was effective at controlling infections caused by A. vannamei P. Phaffia rhodozyma Vibrio hepatarius. mixed into the rearing water increased survival and production of channel catfish (Ictalurus punctatus). (1998) Moriarty (1998) Phianphak et al. L. Vibrio P63. the mortality of larvae decreased in treatments where 107 cfu mlÀ1 Roseobacter sp. (1999) P.. (2004) ? Addition to culture water Scallop larval cultures Pecten maximus Addition to culture water Microalgal cultures Argopecten purpuratus Addition to culture water Chaetoceros gracilis culture Rotifer culture Seawater Commercial product C. (2000) Suminto and Hirayama (1997) Harzevili et al. These strains were identified as Roseobacter spp. brevis. (1999) Riquelme et al. I. monodon P. and an unidentified gram-positive coccus. Vibrio fluvialis. Vibrio ordalii. . Balcazar et al. vannamei P. S11 Bacillus sp. Culture collection L. 2004). and live food Probiotic strain Crustaceans Bacillus sp. / Veterinary Microbiology 114 (2006) 173–186 Table 3 Probiotics considered as biological control agents in aquaculture of crustaceans. previously isolated from the intestine of Atlantic salmon. monodon P.L. P. Vibrio spp. Saccharomyces cerevisiae.. Fish juveniles and adults In experiments performed by Queiroz and Boyd (1998). exiguus. galvana. lactis. vannamei P. Lactobacillus spp. BS107 Alteromona haloplanktis Live food Flavobacterium sp....2. (1998) Ruiz-Ponte et al. monodon Addition to diet Addition to culture water Addition to culture water ´ Balcazar (2003) Gullian et al. strain 27-4 was applied (Hjelm et al. 6. molluscs. Bacillus sp. Lactococcus lactic spp. monodon P. have been beneficial for rainbow trout when these strains were administered as food additives. lutheri Rotifers Chaetoceros muelleri Artemia Artemia nauplii Addition to culture water Addition to culture water Addition to culture water Addition to culture water Addition to culture water Crassostrea gigas Gibson et al. mesenteroides. vannamei P. Carnobacterium sp. (1998) Gomez-Gil et al. Roseobacter spp. Pediococcus acidilactici Pseudoalteromonas. Bacillus P64 Pseudomonas sp. monodon Penaeus vannamei Method of application Reference Addition to diet Addition to culture water Addition to diet Addition to diet 181 Rengpipat et al. Vibrio sp. (2002) Gatesoupe (2002) Villamil et al. 2000). Vibrio fluvialis Molluscs Aeromonas media strain A199 Roseobacter sp. Mesenteroides spp. A strain of Carnobacterium sp..´ J. Lactococcus lactis AR21 V. (2003) Lactobacillus casei. and Pseudoalteromonas. helveticus. gracilis. were not lethal to egg yolk sac turbot larvae and in two of three trials.

the inoculation of Bacillus S11. . a strain isolated from fresh water. 2003). However. A mixture of Lactobacillus spp. Balcazar et al. presented antagonistic activity against Saprolegnia sp.) fed with Enterococcus faecium (strain SF68) were significantly higher than control groups after challenge with Edwardsiella tarda (Chang and Liu. for 6 days before a challenge with V. 2002). harveyi S2 under in vivo conditions of 83. phenol oxidase and antibacterial activity showed declining trends (Alavandi et al. monodon that were challenged by pathogenic luminescent bacterial culture (Rengpipat et al. lactobacilli and enterococci).. subtilis BT23. Of these. 2002). hydrophila. salmonicida. five isolates were inhibitory using its extracellular products to a wide variety of pathogen as A.g. Aeromonas media (strain A199) in culture of eels (Anguilla australis Richardson). Similarly. Also. 2004).. harveyi was controlled by the probiotic effect of Bacillus subtilis BT23 under in vitro and in vivo conditions. One hundred and six bacterial isolates from the stomach and intestine of common clownfish (Amphiprion percula) have been recovered. when shrimp were treated with each of the candidate strains. The probiotic effect in L. respectively. ordalii (Austin et al.. inhibited the growth of Pasteurella piscicida (Sugita et al.3% of Vibrio spp.3% in the 109 cells gÀ1 feed and the 1012 cells gÀ1 feed groups. 1998). 1999). respectively (Nikoskelainen et al. resulted in greater survival of the post-larval P.. isolated from chicken gastrointestinal tracts has improved the growth and survival rates of juvenile P. 2004). Vibrio P63 and Bacillus P64 and achieved inhibition percentages against V. Recently.182 ´ J. From the observations.. they did not help in improving the immune system of shrimp. Crustaceans In a study of tiger shrimp. studies have reported the presence of antifungal effects from various strains of probiotics. supressing the growth of this opportunistic pathogen (Lategan and Gibson.. it was concluded that the turbot intestinal tract and faeces can serve as an enrichment site for V. the estimation of immunological indicators such as haemocyte counts.3. 2001). rhamnosus. the growth of V. a V. V. anguillarum was inhibited by Carnobacterium cells. isolated from shrimp culture ponds. Strains that are generally used as human probiotics (e. fluvialis PM 17 have been selected as candidate probiotics isolated from the gut of farm reared tiger shrimp by the ability to secrete extracellular macromolecule digesting enzymes.. V. 1995). anguillarum and V. It is known that colonization with specific microbiota in the gut may play a role in balancing the intestinal mucosal immune system.. which may contribute to the induction and maintenance of immunological tolerance or to the inhibition of the disregulated responses induced by pathogens in host. alginolyticus strain at 108 cells mlÀ1 was applied in a bath treatment to Atlantic salmon. 60 and 58%.6% in the control to 18. and the use of intestinal bacteria with antagonistic activity against vibrios may be used to reduce the load of fish pathogenic vibrios in turbot hatcheries (Olsson et al. piscicola (Vine et al. alginolyticus and C. salmonicida. harveyi. harveyi at 103–104 cells mlÀ1 for 1 h infection with a 90% reduction in accumulated mortality (Vaseeharan and Ramasamy. Histologic analyses after the colonization and interaction experiment confirmed that the probiotic strains had no pathogenic effect on the host (Gullian et al.. monodon to B. therefore. and to a lesser extent after exposures to V. 2003). Improved disease resistance was observed after exposing juvenile P. The antibacterial abilities of intestinal bacteria isolated from juveniles and larvae of Japanese flounder (Paralichthys olivaceus) have been studied. Possibly these bacteria did not colonize the gut. 2004a). Administration to rainbow trout for 51 days reduced the fish mortality caused by A. V. Recently. salmonicida from 52. In fecal extract from turbot juveniles. 1998). Pseudomonas sp. anguillarum. it has been demonstrated that survival rates of European eels (Anguilla anguilla L. vannamei has been reported using three strains isolated from the hepatopancreas of shrimp. These strains were identified as Vibrio P62. PM 11 and V. A. the growth of pathogenic V. at a density of 106–108 cells mlÀ1. reporting that 53. V. have been considered for studies in fish as a novel and safe treatment in aquaculture. anguillarum. 6.9 and 46. 2002). Also. For example. Experiments revealed that application of the probiotic led to a reduction in mortality after exposures to A. / Veterinary Microbiology 114 (2006) 173–186 since their application reduced significantly the impact of furunculosis by competitive exclusion and enhanced cellular immunity in the fish (Irianto and Austin.L.. monodon when fed these strains for 100 days (Phianphak et al. a saprophytic strain. L. damsela.

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