PROBIOTIC

PROBIOTIC IN AQUACULTURE

INTRODUCTION Aquaculture has become an important economic activity in many countries. In large-scale production facilities, where aquatic animals are exposed to stressful conditions, problems related to diseases and deterioration of environmental conditions often occur and result in serious economic losses. Prevention and control of diseases have led during recent decades to a substantial increase in the use of veterinary medicines. These amounts of antibiotics have exerted a very strong selection pressure towards resistance among bacteria, which have adapted to this situation, mainly by a horizontal and promiscuous flow of resistance genes (SCAN, 2003). Resistance mechanisms can arise one of two ways: chromosomal mutation or acquisition of plasmids. The use of probiotics or beneficial bacteria, which control pathogens through a variety of mechanisms, is increasingly viewed as an alternative to antibiotic treatment. WHAT IS A PROBIOTICS? The term probiotics is generally used to denote bacteria that promote the health of other organisms. Lilley and Stillwell (1965) described them as substances secreted by one microorganism, which stimulated the growth of another. An expert with the Joint Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO), stated that probiotics are live microorganisms, which when consumed in adequate amounts, confer a health benefit for the host (FAO/WHO, 2001). Verschuere et al. (2000) suggested the definition a live microbial adjunct which has a beneficial effect on the host by modifying the host-associated or ambient microbial community, by ensuring improved use of the feed or enhancing its nutritional value, by enhancing the host response towards disease, or by improving the quality of its ambient environment Irianto and Austin (2002) thus a probiotic is an entire or components of a micro-organism that is beneficial to the health of the host.

MECHANISM OF ACTION Probiotic strains have been shown to inhibit pathogenic bacteria both in vitro and in vivo through several different mechanisms. some possible benefits linked to the administering of probiotics have been suggested as: 1. 2. 3. 4. Competitive exclusion of pathogenic bacteria Source of nutrients and enzymatic contribution to digestion Direct uptake of dissolved organic material mediated by the bacteria Enhancement of the immune response against pathogenic microorganisms

5. 6.

Antiviral effects. Interaction with phytoplankton

1. Competitive exclusion Bacterial antagonism is a common phenomenon in nature, therefore, microbial interactions play a major role in the equilibrium between competing beneficial and potentially pathogenic microorganisms. Probiotics competive in three ways:
A. Competition for Adhesion sites B. Competition for Nutrients C. Production of Inhibitory compounds.

A. COMPETITION FOR ADHESION SITES Probiotics Organisms compete with the pathogens for the adhesion sites and food in the gut epithelial surface and finally prevent their colonization. Adhesion capacity and growth on or in intestinal or external mucous has been demonstrated in vitro for fish pathogens like Vibrio anguillarum and Aeromonas hydrophila ( Krovacek, 1987).

B. COMPETITION FOR NUTRIENTS: Probiotics utilizes nutrients which may be consumed by pathogens microbes. Competition for nutrients can play an important role in the composition of microbiota of the intestinal tract or ambient environment of the cultured aquatic organism. C. PRODUCTION OF INHIBITORY COMPOUNDS Probiotic bacteria releases a variety of chemical compounds that are inhibitory to both gram positive and gram negative bacteria. These includes bacteriocines, sideropheres, lysozymes, proteases hydrogen peroxides etc. lactic acid bacteria are known to produce compounds such as bacteriocins that are inhibitory to other microbes. The first report of the existence in seawater of bacteria with an inhibitory effect against a Vibrio sp. has been attributed to Gaixa (1889). In aquaculture, Thalassobacter utilis, has shown inhibitory effects against Vibrio anguillarum. 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.

So competitive exclusion of potential pathogenic bacteria effectively reduces or eliminates the need for antibiotic prophylaxis in intensive larviculture systems. 2. Source of nutrients and enzymatic contribution to digestion It has been suggested that microorganisms have a beneficial effect in the digestive processes of aquatic animals. In fish, it has been reported that Bacteroides and Clostridium sp. have contributed to the host's nutrition, especially by supplying fatty acids and vitamins (Sakata, 1990). Prieur et al (1990) observed that some bacteria may participate in the digestion processes of bivalves by producing extracellular enzymes, such as proteases, lipases, as well as providing necessary growth factors. Dall and Moriarty (1983) reported that 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. 3. Influence on water quality It has been reported that gram positive bacteria especially Bacillus sp. Improve water quality by converting the organic matter back to carbon dioxide into the environment. During the production cycle, high levels of gram-positive bacteria can be minimize the buildup of dissolved and particulate organic carbon. Dalmin et al (2001) reported that use of Bacillus sp. improved water quality, survival and growth rates and increased the health status of juvenile Penaeus monodon and reduced the pathogenic vibrios. 4. Enhancement of the immune response Probiotic stimulate the hosts specific immunity and non specific immunity. Some of the bacteria like Lactic acid bacteria (LAB) increase the resistance to enteric infection. The mechanism of immune stimulation is yet not known, but specific cell wall components or cell layers may act as adjuvants and increase humoral immune response. Sakai et al (1995) demonstrated that oral administration of Clostridium butyricum bacteria to rainbow trout enhanced the resistance of fish to vibriosis, by increasing the phagocytic activity of leucocytes. Nikoskelainen et al. (2003) showed that administration of a lactic acid bacterium Lactobacillus rhamnosus (strain ATCC 53103) at a level of 105 cfu g1 feed, stimulated the respiratory burst in rainbow trout (Oncorhynchus mykiss). 5. Antiviral effects Some bacteria have antiviral effect, but the mechanism which these bacteria do is not known. Kamei et al (1988) reported that strains of Pseudomonas sp., Vibrios sp., Aeromonas sp., and groups of coryneforms isolated from salmonid hatcheries, showed antiviral activity against infectious hematopoietic necrosis virus (IHNV) with more than 50% plaque reduction.

6. Interaction with phytoplankton Probiotics bacteria have a significant algicidal effect on many species of microalgae particularly of red tide plankton. Bacteria antagonistic towards algae would be undesirable in green water larval rearing technique in hatchery where unicellular algae are cultured and added, but would be advantageous when undesired algae species developed in the culture period.

SILENT FEATURE OF PROBIOTICS 1. The probiotic should be non pathogenic and non toxic to the host , 2. It should be accepted by the host, e.g. through ingestion and potential colonization and replication within the host, 3. It should reach the location where the effect is required to take place, 4. It should actually work in vivo as opposed to in vitro findings, 5. It should preferably not contain virulence resistance genes or AB resistance genes. 6. It should be capable of exerting beneficial influence on the host organism ( increase growth and / or disease resistance) 7. it should be capable of surviving and metabolizing in the gut environment ( resistant to low pH and organic acids) 8. It should be capable of remaining viable for a longer period under storage and field conditions. 9. It should be capable of stimulating the immune system. 10. It should be able to modify the microbial component of water such that the pathogenic members are reduced through competitive exclusion. 11. It should be able to efficiently breakdown the organic load with least consumption of dissolved oxygen. 12. It should help to reduce the BOD/COD levels and other obnoxious gases in the sediment. 13. It should be cheap, efficient and easily available.

SELECTION CRITERIA FOR PROBIOTICS Method to select probiotic bacteria for the use in culture system should include the following steps: y COLLECTION OF BACKGROUND INFORMATION Before the start of research on the development of probiotics, the activities about the culture practices and economics of the development should be studied. A close knowledge of the rearing practices used in an aquaculture farm is necessary to determine whether a probiotic application would be feasible or not. ACQUISITION OF PUTATIVE PROBIOTICS The acquisition of good pool of candidate probiotic is of major importance in this process. It is a vital in his process that the choice of the strain is made as a function of the possible role of the probiotics to developed. There is no unequivocal indication that the putative probiotics isolated from the host or from their ambient environment perform better than isolates completely alien to the cultured species or those that originates from a very different habitat. SCREENING OF PUTATIVE PROBIOTICS A common way to screen the candidate probiotics is to perform in vitro antagonism tests in which pathogens are exposed to the candidate probiotic in liquid or in solid medium. Candidate probiotics can be selected based on the production of inhibitory compounds like Bacteriocines, siderophores, or when in competition of nutrients. This is done with extreme precautions. EVALUATION OF PATHOGENICITY OF SELECTED STRAINS Probiotic should not be pathogenic to the host and this should be confirmed prior to acceptance. Therefore the host must be challenged under stressed and non stressed conditions. This can be done by adding probiotic to the cultured water. IN VIVO EVALUATION The effect of candidate probiotic should also be evaluated in vivo, in which the candidate species is introduced to the host under culture and then monitoring growth, survival and physic-chemical parameters.

PREPARATION OF PROBIOTIC FOR AQUACULTURE 1. SCREENING OF POTENTIAL PROBIOTIC It is done to isolate the microbe from the culture and enhance its natural effects, favoring their growth by adding some nutrients or distributing large quantities of them in culture.

2. SUITABLE CAREER OF PROBITIC It is done along with feed in pelleted, encapsulated and free concentrated forms to reach the gut in sufficient numbers and to see see the viability of these for a longer period.

TYPES OF PROBIOTIC Conventionally probiotics are applied through feed, but now a day they are also being applied to the pond water or sediments. Based on their mode of action they are classified as 1. Feed probiotics 2. Pond probiotics FEED PROBIOTICS Feed probiotics are those that are administered through feed. These finally find their way into the gut or gastrointestinal tracts of the cultured organism. On reaching the gastro-intestinal tract the feed probiotics act thougha. Suppression of bacterial pathogens b. Production of anti-bacterial compounds like organic acids, H2O2 and antibiotics by anaerobic bacteria results in competitive exclusion of pathogenic strains from the guts of the host. c. Production of special metabolites like vitamin B by many probiotic strain leads to better nutrient utilization and health condition. d. Immunostimulation- several strains of Lactobacilli have some potential role in augmenting the immune system of host. e. Reduction of the potential availability of oxygen to pathogen. f. Detoxification of the metabolites produced by intestinal pathogens.

ADVANTAGES OF FEED POBIOTICS 1. Promotion of growth 2. Improved food conversion 3. Prevention of intestinal disorder 4. Pre-digestion of anti nutritional factor 5. Avoidance of residual effect of antibiotic in tissue.

POND PROBIOTICS Health of aquatic organism deteriorates rapidly with increasing environmental stress like low DO, accumulation of ionized ammonia, nitrite, anoxic conditions and H2 S in the sediments. So

many microbial supplements can be used to mitigate such conditions which prevent their establishment in ponds. Pond probiotics basically settles on the botton sediments and act from the bottom. ACTTIONS OF POND PROBIOTIC 1. Biocontrol probiotic limit or eliminate the pathogenic biota through antagonistic properties. 2. Bioremediation - probiotic maintain water quality through reduction in BOD or COD level. The responsible bacteria have capacity to breakdown complex organic matter to simpler units and are also capable of oxidizing toxic element from water and render them harmless.

PROBIOTIC STRAINS STUDIED IN AQUACULTURE 1. FISH EGGS AND LARVAE The spawning and early life stages of fish larvae may have profound implications for the dynamics of microbial communities. These communities can also be influenced by inorganic and organic compounds. Immediately after hatching process, fish larvae come into contact with their immediate environment that provides colonization by a wide variety of microorganism. The composition of microbiota is influenced by many factors including the availability of nutrients, animal physiology and immunological factors. Under normal conditions, 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. During the initial feeding period, it is possible to manipulate the establishment of an artificial dominance of a determined group of bacteria in the fish-associated microbiota by adding a specific strain. Example, . A significant increase in the mean weight and survival rate of turbot larvae (S. maximus) fed rotifers enriched in lactic acid bacteria and these strains provided a significant protection against a pathogenic Vibrio compared to control larvae (Gatesoupe, 1994). 2. FISH JUVENILES AND ADULT It is found that a commercially prepared bacterial mixture of Bacillus spp. mixed into the rearing water increased survival and production of channel catfish (Ictalurus punctatus). A strain of Carnobacterium sp. previously isolated from the intestine of Atlantic salmon, was effective at controlling infections caused by A. salmonicida, Vibrio ordalii, and Yersinia ruckeri in fry and fingerling salmonids, applied at 5 107 cells g1 of feed .

Cultures of Aeromonas hydrophila, Vibrio fluvialis, Carnobacterium sp. and an unidentified gram-positive coccus, have been beneficial for rainbow trout when these strains were administered as food additives, since their application reduced significantly the impact of furunculosis by competitive exclusion and enhanced cellular immunity in the fish. It has been reported that the presence of antifungal effects from various strains of probiotics. For example, a strain isolated from fresh water, Aeromonas media (strain A199) in culture of eels (Anguilla australis Richardson), presented antagonistic activity against Saprolegnia sp., supressing the growth of this opportunistic pathogen. 3. CRUSTACEAN In a study of tiger shrimp, the inoculation of Bacillus S11, a saprophytic strain, resulted in greater survival of the post-larval P. monodon that were challenged by pathogenic luminescent bacterial culture. A mixture of Lactobacillus spp. isolated from chicken gastrointestinal tracts has improved the growth and survival rates of juvenile P. monodon when fed these strains for 100 days

CONSTRAINTS OF PROBIOTIC USE The major constraints of probiotic are 1. LEACHING Probiotic strains used in the feed may not be available to host due to leaching, therefore standardization of dose is essential. 2. SURVIVAL Many bacterial strains not survive in feed preparation and they lose their viability when exposed to high temperature and pressure of the extruder. 3. HIGH ORGANIC LOAD - High organic load in the bottom sediments may reduce the efficacy of the bioremediator. 4. DOSAGES Depending on the water and sediment status exact dosages need to be calculated for every set of different conditions.

CONCLUSION Probiotic have been successfully used in aquaculture to enhance both internal and external microbial
environment. However a great deal of research is needed to make it successfully proposition. First of all it is necessary to understand the nature of competition between species or strain. Secondly fate of probiotic organism in the rearing medium and in gastrointestinal tract should be answered. Thirdly there is a need for future studies to determine the best possible method of adminstration of probiotic. The mechanism by which the bacterial treatment influences the fish survival cannot be explained from data collected therefore further initiatives to define the potential benefits of these treatecosystem is essential.ments to aquaculture production and further derterminig their mechanism of action in pond

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SELECTION OF PROBIOTICS AS BIOCONTROL AGENTS IN AQUACULTURE.