Thursday, August 4, 2016

Probiotic Bacteria as Biological Control Agents in Aquaculture

There is an urgent need in aquaculture to develop microbial control strategies, since disease outbreaks are recognized as important constraints to aquaculture production and trade and since the development of antibiotic resistance has become a matter of growing concern. One of the alternatives to antimicrobials in disease control could be the use of probiotic bacteria as microbial control agents. This review describes the state of the art of probiotic research in the culture of fish, crustaceans, mollusks, and live food, with an evaluation of the results obtained so far. A new definition of probiotics, also applicable to aquatic environments, is proposed, and a detailed description is given of their possible modes of action, i.e., production of compounds that are inhibitory toward pathogens, competition with harmful microorganisms for nutrients and energy, competition with deleterious species for adhesion sites, enhancement of the immune response of the animal, improvement of water quality, and interaction with phytoplankton. A rationale is proposed for the multistep and multidisciplinary process required for the development of effective and safe probiotics for commercial application in aquaculture. Finally, directions for further research are discussed.

Aquaculture of finfish, crustaceans, mollusks, and algal plants is one of the fastest-growing food-producing sectors, having grown at an annual rate of almost 10% from 1984 to 1995 compared with 3% for livestock meat and 1.6% for capture fisheries production.

Disease outbreaks are being increasingly recognized as a significant constraint on aquaculture production and trade, affecting the economic development of the sector in many countries. For instance, disease is now considered to be the limiting factor in the shrimp culture subsector. So far, conventional approaches, such as the use of disinfectants and antimicrobial drugs, have had limited success in the prevention or cure of aquatic disease. Furthermore, there is a growing concern about the use and, particularly, the abuse of antimicrobial drugs not only in human medicine and agriculture but also in aquaculture. The massive use of antimicrobials for disease control and growth promotion in animals increases the selective pressure exerted on the microbial world and encourages the natural emergence of bacterial resistance (World Health Organization antimicrobial resistance fact sheet 194, Not only can resistant bacteria proliferate after an antibiotic has killed off the other bacteria, but also they can transfer their resistance genes to other bacteria that have never been exposed to the antibiotic. The subtherapeutic (prophylactic) use of antibiotics related to those used in human medicine or the use of any antimicrobial agent known to select for cross-resistance to antimicrobials used in human medicine could pose a particularly significant hazard to human health.

According to the World Health Organization (fact sheet 194 web site), much needs to be done to reduce the overuse and inappropriate use of antimicrobials. The emphasis in disease management should be on prevention, which is likely to be more cost-effective than cure. This may lead to less reliance on the use of chemicals (antimicrobials, disinfectants, and pesticides), which largely treat the symptoms of the problem and not the cause.

Several alternative strategies to the use of antimicrobials in disease control have been proposed and have already been applied very successfully in aquaculture. The use of antimicrobial drugs in a major producing country such as Norway has dropped from approximately 50 metric tons per year in 1987 to 746.5 kg in 1997, measured as active components. During the same time, the production of farmed fish in Norway increased approximately from 5 × 104 to 3.5 × 105 metric tons. The dramatic decrease observed in the consumption of antimicrobial agents is mainly due to the development of effective vaccines which illustrates very well the potential effectiveness of the procedure. Enhancing the nonspecific defense mechanisms of the host by immunostimulants, alone or in combination with vaccines, is another very promising approach. Third, Yasuda and Taga already anticipated in 1980 that bacteria would be found to be useful both as food and as biological control agents of fish disease and activators of the rate of nutrient regeneration in aquaculture. Vibrio alginolyticus has been employed as a probiotic in many Ecuadoran shrimp hatcheries since late 1992. As a result, hatchery down time was reduced from approximately 7 days per month to less than 21 days annually, while production volumes increased by 35%. The overall antibiotic use was decreased by 94% between 1991 and 1994. The addition of probiotics is now also common practice in commercial shrimp hatcheries in Mexico. According to Browdy, one of the most significant technologies that has evolved in response to disease control problems is the use of probiotics. Considering the recent successes of these alternative approaches, the Food and Agriculture Organization of the United Nations defined the development of affordable yet efficient vaccines, the use of immunostimulants and nonspecific immune enhancers, and the use of probiotics and bioaugmentation for the improvement of aquatic environmental quality as major areas for further research in disease control in aquaculture. The results of this research will undoubtedly help to reduce chemical and drug use in aquaculture and will make aquaculture products more acceptable to consumers.

This review aims to provide an overview of the work done on bacteria as biological control agents for aquaculture environments, with a critical evaluation of the results obtained so far and a detailed description of the possible modes of action involved. Furthermore, a rationale for the search for probiotics is presented and directions for further research are proposed.

As new findings emerged, several definitions of probiotics have been proposed. Fuller  gave a precise definition of probiotics which is still widely referred to, i.e., a live microbial feed supplement which beneficially affects the host animal by improving its intestinal balance.

Historically, the interest has centered on terrestrial organisms, and the term “probiotic” inevitably referred to gram-positive bacteria associated with the genusLactobacillus. The application of the definition proposed by Fuller in aquaculture, however, requires some considerations. Similarly to humans and terrestrial animals, it can be assumed in aquaculture that the intestinal microbiota does not exist as entity by itself but that there is a constant interaction with the environment and the host functions. Many researchers have already investigated the relationship of the intestinal microbiota to the aquatic habitat or food. Cahill summarized the results of these investigations on fishes, giving evidence that the bacteria present in the aquatic environment influence the composition of the gut microbiota and vice versa. The genera present in the intestinal tract generally seem to be those from the environment or the diet which can survive and multiply in the intestinal tract. However, it can be claimed that in aquaculture systems the immediate ambient environment has a much larger influence on the health status than with terrestrial animals or humans.

Indeed, the host-microbe interactions are often qualitatively as well as quantitatively different for aquatic and terrestrial species. In the aquatic environment, hosts and microorganisms share the ecosystem. By contrast, in most terrestrial systems, the gut represents a moist habitat in an otherwise water-limitted environment. In some sense, microbes in an aquatic environment have the choice of living in association with the potential host (intestinal tract, gills, or skin) or not, while in the terrestrial environment, appreciable activity may be limited to aquatic niches such as those provided by the guts of host animals.

Much more than terrestrial animals, aquatic farmed animals are surrounded by an environment that supports their pathogens independently of the host animals, and so (opportunistic) pathogens can reach high densities around the animal. Surrounding bacteria are continuously ingested either with the feed or when the host is drinking. This is especially the case with filter feeders, which ingest bacteria at a high rate from the culture water, causing a natural interaction between the microbiota of the ambient environment and the live food.

While probiotic research in aquaculture focused in the beginning on fish juveniles, more attention has recently been given to larvae of fish and shellfish and to live food organisms Terrestrial animals (mammals) inherit an important part of the initially colonizing bacteria through contact with the mother, while aquatic species usually spawn axenic eggs in the water, without further contact with the parents. This allows ambient bacteria to colonize the egg surface. Furthermore, freshly hatched larvae or newborn animals do not have a fully developed intestinal system and have no microbial community in the intestinal tract, on the gills, or on the skin. Because the early stages of aquatic larvae depend for their primary microbiota partly on the water in which they are reared, the properties of the bacteria in the ambient water are of the utmost importance.

Overview of literature reports dealing with probiotics as biological control agents in aquaculture

It was stated above that the interaction between the microbiota, including probiotics, and the host is not limited to the intestinal tract. Probiotic bacteria could also be active on the gills or the skin of the host but also in its ambient environment. The intensive interaction between the culture environment and the host in aquaculture implies that a lot of probiotics are obtained from the culture environment and not directly from feed, as stipulated by the definition of Fuller.

Therefore, the following modified definition is proposed, which allows a broader application of the term “probiotic” and addresses to the objections made earlier. A probiotic is defined as 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.

Based on this definition, probiotics may include microbial adjuncts that prevent pathogens from proliferating in the intestinal tract, on the superficial structures, and in the culture environment of the cultured species, that secure optimal use of the feed by aiding in its digestion, that improve water quality, or that stimulate the immune system of the host. Bacteria delivering essential nutrients to the host (single-cell protein) without being active in the host or without interacting with other bacteria, with the environment of the host, or with the host itself are not included in the definition. Although probiotics may also contribute substantially to the health and zootechnical performance in a nutritional way and although it is sometimes impossible to separate feeding of aquatic organisms from environmental control, this review is limited to the use of probiotics as biological control agents in aquaculture.

Aquaculture practices such as discontinuous culture cycles, disinfection or cleaning of ponds or tanks prior to stocking, and sudden increases in nutrients due to exogenous feeding generally do not provide appropriate environments for the establishment of stable microbial communities. Therefore, it is very unlikely that under intensive rearing conditions a stable microbial community can be achieved. In the development of these microbial communities, one should consider both deterministic and stochastic factors. Deterministic factors have a well-defined dose-response relationship. For a given value of a stochastic factor, a probability range of responses can occur. Deterministic factors influencing the microbial development in aquaculture systems include salinity, temperature, oxygen concentration, and quantity and quality of the feed. These combined environmental factors create a habitat in which a selected and well-defined range of microbes is able to proliferate (“the environment selects” axiom). The development of a microbial community in aquaculture systems is, however, also influenced by stochastic phenomena: chance favors organisms which happen to be in the right place at the right time to enter the habitat and to proliferate if the conditions are suitable.

This theoretical concept has been experimentally supported by Verschuere et al., who monitored the community-level physiological profiles of the emerging microbial communities in the culture water of Artemia juveniles in three identical culture series. Although completely identical from the zootechnical point of view, the culture water of the three series showed clearly distinct microbial communities developing in the first days of the experiment. The same concept may be valid for the microbial communities developing in the culture water and on the inner and outer surfaces of eggs and larval organisms. Obviously, due to the heterogeneity of the microbial distribution in the air and water, in feeds, and on surfaces, the stochastic factors are very important in the colonization of aquacultural environments.

The idea that both environmental conditions and chance influence the emergence of microbial communities opens opportunities for the concept of probiotics as biological conditioning and control agents. Instead of allowing spontaneous primary colonization of the rearing water by bacteria accidentally present, the water could be preemptively colonized by the addition of probiotic bacteria, since it is generally recognized that preemptive colonization may extend the reign of pioneer organisms. It is suggested that in the case of preemptive colonization of rearing environments with emerging microbial communities, a single addition of a probiotic culture may suffice to achieve colonization and persistence in the host and/or in its ambient environment, provided that the probiotic cultures are well adapted to the prevailing environmental conditions. When the host or its environment already carries a well-established and stable microbial community, it is much more probable that the probiotic will have to be supplied on a regular basis to achieve and maintain its artificial dominance.

It is therefore a pertinent question whether it is possible to modify the composition of a microbial community in the field by the exogenous addition of a probiotic. This is particularly important when a long-term exposure is required for the probiotic effect. It is not easy to answer this question, since the literature does not provide real evidence for this in aquacultural practices. Nevertheless, some assumptions can be made when referring to work done with lactic acid bacteria. Although lactic acid bacteria are not dominant in the normal intestinal microbiota of larval or growing fish, several trials have been done to induce an artificial dominance of lactic acid bacteria in fish fry. The addition of high doses of lactic acid bacteria to established microbial communities of fish juveniles provoked a temporary change in the composition of the intestinal microbial community. Within a few days after the intake had stopped, however, the added strains showed a sharp decrease and were lost from the gastrointestinal tract in most of the fish. Several reports describe bacteria firmly attached to the intestinal mucosa, and it is now accepted that fish contain a specific intestinal microbiota that becomes established at the juvenile stage or after metamorphosis. Unless the host has been exposed to a limited range of microorganisms in its development, it is improbable that a single exogenous addition of a probiotic to an established microbial community will result in long-term dominant colonization. This seems to be particularly the case when bacterial species are used which do not belong to the normal dominant intestinal microbiota of the cultured species or its particular development stage. In such cases, it is necessary to supply the probiotic on a regular basis if a continuous colonization at high densities is required. See Full article at

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