probiotic bacteria as biological control agents

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INTRODUCTION

Aquaculture of finfish, crustaceans, mollusks, and algalplants is one of the fastest-growing food-producing sectors,having grown at an annual rate of almost 10% from 1984 to1995 compared with 3% for livestock meat and 1.6% for cap-ture fisheries production (97).

Disease outbreaks are being increasingly recognized as asignificant constraint on aquaculture production and trade,affecting the economic development of the sector in manycountries. For instance, disease is now considered to be thelimiting factor in the shrimp culture subsector (65, 124). So far,conventional approaches, such as the use of disinfectants andantimicrobial drugs, have had limited success in the preventionor cure of aquatic disease (124). Furthermore, there is a grow-ing concern about the use and, particularly, the abuse of anti-microbial drugs not only in human medicine and agriculturebut also in aquaculture. The massive use of antimicrobials fordisease control and growth promotion in animals increases theselective pressure exerted on the microbial world and encour-ages the natural emergence of bacterial resistance (WorldHealth Organization antimicrobial resistance fact sheet 194,http://www.who.int/inf-fs/en/fact194.html). Not only can resis-tant bacteria proliferate after an antibiotic has killed off theother bacteria, but also they can transfer their resistance genesto other bacteria that have never been exposed to the antibi-otic. The subtherapeutic (prophylactic) use of antibiotics re-lated to those used in human medicine or the use of anyantimicrobial agent known to select for cross-resistance to an-timicrobials used in human medicine could pose a particularlysignificant hazard to human health (146).

According to the World Health Organization (fact sheet 194web site), much needs to be done to reduce the overuse andinappropriate use of antimicrobials. The emphasis in diseasemanagement should be on prevention, which is likely to bemore cost-effective than cure. This may lead to less reliance on

the use of chemicals (antimicrobials, disinfectants, and pesti-cides), which largely treat the symptoms of the problem andnot the cause (92).

Several alternative strategies to the use of antimicrobials indisease control have been proposed and have already beenapplied very successfully in aquaculture. The use of antimicro-bial drugs in a major producing country such as Norway hasdropped from approximately 50 metric tons per year in 1987 to746.5 kg in 1997, measured as active components. During thesame time, the production of farmed fish in Norway increasedapproximately from 5 104 to 3.5 105 metric tons. Thedramatic decrease observed in the consumption of antimicro-bial agents is mainly due to the development of effective vac-cines (66, 124), which illustrates very well the potential effec-tiveness of the procedure. Enhancing the nonspecific defense

mechanisms of the host by immunostimulants, alone or incombination with vaccines, is another very promising approach(96, 111). Third, Yasuda and Taga (148) already anticipated in1980 that bacteria would be found to be useful both as foodand as biological control agents of fish disease and activators ofthe rate of nutrient regeneration in aquaculture. Vibrio algino-lyticus has been employed as a probiotic in many Ecuadoranshrimp hatcheries since late 1992 (49). As a result, hatcherydown time was reduced from approximately 7 days per monthto less than 21 days annually, while production volumes in-creased by 35%. The overall antibiotic use was decreased by94% between 1991 and 1994. The addition of probiotics is nowalso common practice in commercial shrimp hatcheries inMexico (101). According to Browdy (14), one of the mostsignificant technologies that has evolved in response to disease

control problems is the use of probiotics. Considering the re-cent successes of these alternative approaches, the Food andAgriculture Organization of the United Nations (124) definedthe development of affordable yet efficient vaccines, the use ofimmunostimulants and nonspecific immune enhancers, and theuse of probiotics and bioaugmentation for the improvement ofaquatic environmental quality as major areas for further re-

search in disease control in aquaculture. The results of thisresearch will undoubtedly help to reduce chemical and druguse in aquaculture and will make aquaculture products moreacceptable to consumers.

This review aims to provide an overview of the work done onbacteria as biological control agents for aquaculture environ-ments, with a critical evaluation of the results obtained so farand a detailed description of the possible modes of actioninvolved. Furthermore, a rationale for the search for probioticsis presented and directions for further research are proposed.

DEFINITION OF PROBIOTICS

As new findings emerged, several definitions of probioticshave been proposed. Fuller (30) gave a precise definition ofprobiotics which is still widely referred to, i.e., a live microbialfeed supplement which beneficially affects the host animal byimproving its intestinal balance.

Historically, the interest has centered on terrestrial organ-isms, and the term probiotic inevitably referred to gram-positive bacteria associated with the genus Lactobacillus. Theapplication of the definition proposed by Fuller (30) in aquac-ulture, however, requires some considerations. Similarly tohumans and terrestrial animals (56), it can be assumed inaquaculture that the intestinal microbiota does not exist asentity by itself but that there is a constant interaction with theenvironment and the host functions. Many researchers havealready investigated the relationship of the intestinal micro-biota to the aquatic habitat or food. Cahill (17) summarized

the results of these investigations on fishes, giving evidencethat the bacteria present in the aquatic environment influencethe composition of the gut microbiota and vice versa. Thegenera present in the intestinal tract generally seem to be thosefrom the environment or the diet which can survive and mul-tiply in the intestinal tract (17). However, it can be claimed thatin aquaculture systems the immediate ambient environmenthas a much larger influence on the health status than withterrestrial animals or humans.

Indeed, the host-microbe interactions are often qualitativelyas well as quantitatively different for aquatic and terrestrialspecies. In the aquatic environment, hosts and microorganismsshare the ecosystem. By contrast, in most terrestrial systems,the gut represents a moist habitat in an otherwise water-limit-ted environment. In some sense, microbes in an aquatic envi-

ronment have the choice of living in association with the po-tential host (intestinal tract, gills, or skin) or not, while in theterrestrial environment, appreciable activity may be limited toaquatic niches such as those provided by the guts of hostanimals (53).

Much more than terrestrial animals, aquatic farmed animalsare surrounded by an environment that supports their patho-gens independently of the host animals, and so (opportunistic)pathogens can reach high densities around the animal (73).Surrounding bacteria are continuously ingested either with thefeed or when the host is drinking. This is especially the casewith filter feeders, which ingest bacteria at a high rate from theculture water, causing a natural interaction between the mi-crobiota of the ambient environment and the live food.

While probiotic research in aquaculture focused in the be-

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ginning on fish juveniles, more attention has recently beengiven to larvae of fish and shellfish and to live food organisms(Table 1). Terrestrial animals (mammals) inherit an importantpart of the initially colonizing bacteria through contact with themother, while aquatic species usually spawn axenic eggs in the water, without further contact with the parents. This allowsambient bacteria to colonize the egg surface. Furthermore,

freshly hatched larvae or newborn animals do not have a fullydeveloped intestinal system and have no microbial communityin the intestinal tract, on the gills, or on the skin. Because theearly stages of aquatic larvae depend for their primary micro-biota partly on the water in which they are reared (17, 50, 102),the properties of the bacteria in the ambient water are of theutmost importance (115).

It was stated above that the interaction between the micro-biota, including probiotics, and the host is not limited to theintestinal tract. Probiotic bacteria could also be active on thegills or the skin of the host but also in its ambient environment.The intensive interaction between the culture environment andthe host in aquaculture implies that a lot of probiotics areobtained from the culture environment and not directly fromfeed, as stipulated by the definition of Fuller (30).

Therefore, the following modified definition is proposed,which allows a broader application of the term probiotic andaddresses to the objections made earlier. A probiotic is definedas a live microbial adjunct which has a beneficial effect on thehost by modifying the host-associated or ambient microbialcommunity, by ensuring improved use of the feed or enhancingits nutritional value, by enhancing the host response towardsdisease, or by improving the quality of its ambient environment.

Based on this definition, probiotics may include microbialadjuncts that prevent pathogens from proliferating in the in-testinal tract, on the superficial structures, and in the cultureenvironment of the cultured species, that secure optimal use ofthe 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 withother bacteria, with the environment of the host, or with thehost itself are not included in the definition. Although probi-otics may also contribute substantially to the health andzootechnical performance in a nutritional way and although itis sometimes impossible to separate feeding of aquatic organ-isms from environmental control, this review is limited to theuse of probiotics as biological control agents in aquaculture.

FUNDAMENTAL QUESTION: IS IT POSSIBLE TOMANIPULATE MICROBIAL COMMUNITIES?

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 gen-erally do not provide appropriate environments for the estab-lishment of stable microbial communities. Therefore, it is veryunlikely that under intensive rearing conditions a stable micro-bial community can be achieved (116). In the development ofthese microbial communities, one should consider both deter-ministic and stochastic factors (74, 139). Deterministic factorshave a well-defined dose-response relationship. For a givenvalue of a stochastic factor, a probability range of responsescan occur. Deterministic factors influencing the microbial development in aquaculture systems include salinity, tempera-ture, oxygen concentration, and quantity and quality of thefeed. These combined environmental factors create a habitat inwhich a selected and well-defined range of microbes is able toproliferate (the environment selects axiom). The develop-

ment of a microbial community in aquaculture systems is, how-ever, also influenced by stochastic phenomena: chance favorsorganisms which happen to be in the right place at the righttime to enter the habitat and to proliferate if the conditions aresuitable (74).

This theoretical concept has been experimentally supportedby Verschuere et al. (139), who monitored the community-

level physiological profiles of the emerging microbial commu-nities in the culture water of Artemia juveniles in three iden-tical culture series. Although completely identical from thezootechnical point of view, the culture water of the three seriesshowed clearly distinct microbial communities developing inthe first days of the experiment. The same concept may be validfor the microbial communities developing in the culture waterand on the inner and outer surfaces of eggs and larval organ-isms. Obviously, due to the heterogeneity of the microbialdistribution in the air and water, in feeds, and on surfaces, thestochastic factors are very important in the colonization ofaquacultural environments.

The idea that both environmental conditions and chanceinfluence the emergence of microbial communities opens op-portunities for the concept of probiotics as biological condi-tioning and control agents. Instead of allowing spontaneousprimary colonization of the rearing water by bacteria acciden-tally present, the water could be preemptively colonized by theaddition of probiotic bacteria, since it is generally recognizedthat preemptive colonization may extend the reign of pioneerorganisms (1). It is suggested that in the case of preemptivecolonization of rearing environments with emerging microbialcommunities, a single addition of a probiotic culture may suf-fice to achieve colonization and persistence in the host and/orin its ambient environment, provided that the probiotic cul-tures are well adapted to the prevailing environmental condi-tions. When the host or its environment already carries a well-established and stable microbial community, it is much moreprobable that the probiotic will have to be supplied on a reg-

ular basis to achieve and maintain its artificial dominance.It is therefore a pertinent question whether it is possible tomodify the composition of a microbial community in the fieldby the exogenous addition of a probiotic. This is particularlyimportant when a long-term exposure is required for the pro-biotic effect. It is not easy to answer this question, since theliterature does not provide real evidence for this in aquacul-tural practices. Nevertheless, some assumptions can be made when referring to work done with lactic acid bacteria (103).Although lactic acid bacteria are not dominant in the normalintestinal microbiota of larval or growing fish (103), severaltrials have been done to induce an artificial dominance of lacticacid bacteria in fish fry (32, 37, 43, 44, 59, 123). The addition ofhigh doses of lactic acid bacteria to established microbial com-munities of fish juveniles provoked a temporary change in the

composition of the intestinal microbial community. Within afew days after the intake had stopped, however, the addedstrains showed a sharp decrease and were lost from the gas-trointestinal tract in most of the fish (59, 103). Several reportsdescribe bacteria firmly attached to the intestinal mucosa (102,112, 122), and it is now accepted that fish contain a specificintestinal microbiota that becomes established at the juvenilestage or after metamorphosis. Unless the host has been ex-posed to a limited range of microorganisms in its development,it is improbable that a single exogenous addition of a probioticto an established microbial community will result in long-termdominant colonization. This seems to be particularly the casewhen bacterial species are used which do not belong to thenormal dominant intestinal microbiota of the cultured speciesor its particular development stage. In such cases, it is neces-

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TABLE 1. Overview of literature reports dealing with probiotics as biological control agents in aquaculture

Putative probiotic Origina ObservationsMethod of

administrationaSuggested mode

of actionaReference(s)

Fish eggs and LarvaeSeveral strains

(unidentified)Eggs of cod

and halibutFailure of the strains to prevent

the adherence of environ-mental bacteria to cod eggs

Bathing in bacte-rial suspension

Antagonism 51

Vibrio salmonicida-like andLactobacillus plantarum Fish Increase of survival of halibutlarvae 2 weeks after hatching Addition to cul-ture water Immunostimulation 84

Bacillus strain IP5832spores (Paciflor 9)

? Increase of weight of turbotlarvae when fed spore-fedrotifers; decrease of mortality

when challenged with anopportunistic Vibrionaceaemember

Addition torotifer diet

Antagonism and/orimproved nutri-tional value ofthe rotifers

35

Streptococcus lactis andLactobacillus bulgaricus

? Increase of survival of turbotlarvae 17 days after hatching

Enrichment ofrotifers andArtemia

? 32

Lactobacillus orCarnobacterium

Rotifers (Brachi-onus plica-tilis)

Decrease of mortality of turbotlarvae challenged with apathogenic Vibrio sp.

Enrichment ofrotifers

Antagonism and/orimproved nutri-tional value ofthe rotifers

37

Vibrio pelagius Copepod-fedturbot larvae

Decrease (?) of mortality ofturbot larvae challenged with A. caviae

Addition to cul-ture water

? 104

Strain E (Vibrioalginolyticus-alike) Healthy turbotlarvae Decrease of mortality of turbotlarvae challenged with apathogenic Vibrio strain P;also, the growth rate of thelarvae may be increased

Enrichment ofrotifers Competition foriron 38

Microbially matured water Increase of initial growth rateof turbot and halibut larvae

As culture water ? 115, 135

Fish juveniles and AdultsLyophilized

Carnobacterium divergensAtlantic salmon

intestinesIncrease (!) of mortality of

Atlantic salmon fry chal-lenged with cohabitantsinfected with A. salmonicida

Addition to diet 43

LyophilizedCarnobacterium divergens

Atlantic salmonintestines

Decrease of mortality of Atlan-tic cod fry when challenged

with a pathogenic V. anguilla-rum strain

Addition to diet ? 45

LyophilizedCarnobacterium divergens

Atlantic salmonintestines

Decrease of mortality of Atlan-tic cod fry challenged with apathogenic V. anguillarumstrain 12 days after the infec-tion; 4wk after the infection,however, the same mortalityas in the control was reached

Additon to diet Antagonism 44

Carnobacterium strain K1 Atlantic salmonintestines

Growth inhibition of V. anguil-larum and A. salmonicidain fish intestinal mucus andfecal extracts (no in vivo test)

Antagonism 59

Carnobacterium Atlantic salmonintestines

Growth inhibition of V. anguil-larum in turbot fecal extracts(no in vivo test)

Antagonism 85

Fluorescent pseudomonadF19/3

Fish mucus Decrease of mortality of Atlan-tic salmon presmolts chal-lenged with stress-inducibleA. salmonicida infection


Competition foriron

117

Pseudomonas fluorescensAH2

Iced Lake Vic-torian Nileperch

Decrease of mortality of rain-bow trout juveniles chal-lenged with a pathogenic

V. anguillarum

Addition to cul-ture waterand/or bathing

in bacterialsuspension

Competition foriron

48

Vibrio alginolyticus Commercialshrimphatchery inEcuador

Decrease of mortality of Atlan-tic salmon juveniles chal-lenged with a pathogenicA. salmonicida, V. anguilla-rum, and V. ordalii


Antagonism 5

Bacillus megaterium,B. polymyxa, B. licheni-formis, 2 strains ofB. subtilis (Biostart)

? Increase of survival and netproduction of channel catfish

Addition to pondwater

? 95

Spray-dried Tetraselmissuecica (unicellular alga)

? Decrease of mortality of Atlan-tic salmon juveniles chal-lenged with several patho-gens; alga was effectiveprophylactically as well astherapeutically

Addition to diet Antagonism 2

Continued on following page



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sary to supply the probiotic on a regular basis if a continuouscolonization at high densities is required.

RECENT FINDINGS

General Considerations

It was suggested in 1980 by Yasuda and Taga (148) thatbacteria would be found to be useful not only as food but alsoas biological controllers of fish disease and activators of nutri-ent regeneration. Only in the late 1980s did the first publica-

tions on biological control in aquaculture emerge, and sincethen the research effort has continually increased. Generally,probiotics are applied in the feed or added to the culture tankor pond as preventive agents against infection by pathogenicbacteria, although nutritional effects are also often attributedto probiotics, especially for filter feeders.

Most probiotics proposed as biological control agents inaquaculture belong to the lactic acid bacteria (Lactobacillus,Carnobacterium, etc.), to the genus Vibrio (Vibrio alginolyticus,etc.), to the genus Bacillus, or to the genus Pseudomonas,although other genera or species have also been mentioned.An overview of the published results is given in Table 1.

TABLE 1Continued

Putative probiotic Origina ObservationsMethod of

administrationaSuggested mode

of actionaReference(s)

CrustaceansBacillus strain S11 Penaeus mono-

don or mud

and waterfrom shrimpponds

Increase of mean weight andsurvival of P. monodon larvae

and postlarvae; decrease ofmortality after challenge withthe pathogen V. harveyi D331

Addition to diet Antagonism 99; Rengpipat andRukpratanporn,

abstract

Vibrio alginolyticus Pacific Oceanseawater

Increase of survival and weightof L. vannamei postlarvae;decreased observation ofV. parahaemolyticus in theshrimps


Antagonism 33

Bacillus ? Increase of survival of penacidshrimps; decrease of lumi-nous Vibrio densities

Addition to pondwater

Antagonism 73, 74

Strain PM-4 and/or NS-110 Soil Increase of survival ofP. mono-don and P. trituberculatuslarvae; decrease of Vibriodensities


Antagonism/foodsource for larvae

6769, 83

Strain BY-9 Coastal sea-water

Increase of survival of P. mono-don larvae; decrease of Vibriodensities


? Sugama andTsumura,abstract

Bivalve mollusksVibrio strain 11 Microalgae ina scallophatchery

Decrease of mortality of scalloplarvae challenged with apathogenic V. anguillarum-like strain


Antagonism 105

Aeromonas media A 199 ? Decrease of mortality and sup-pression of the pathogen ofPacific oyster larvae whenchallenged with a pathogenicV. tubiashii


Antagonism 42

Live foodAlgaeSeveral strains Turbot larvae Growth stimulation of P. lutheri Addition to cul-

ture water? 75

Flavobacterium sp. Chaetocerosgracilis massculture

Improved growth characteristicsof C. gracilis, I. galbana, and P. lutheri


? 128

Strain SK-05 Skeletonemacostatumculture

Inhibition of the growth ofV. alginolyticus in Skele-tonema costatum culture


Competition forresources

101

Live foodRotifersLactobacillus plantarum ? Inhibition of the growth of a

fortuitous A. salmonicidastrain in rotifer culture

Addition to diet Antagonism 36

Lactococcus lactis AR21 Rotifer culture Counteraction of rotifer growthinhibition when challenged

with V. anguillarum


? 54

Several strains Rotifer culture Increase of the specific growthrate of rotifer culture


108

Live foodArtemiaVibrio alginolyticus C14 ? Decrease of mortality of

Artemia nauplii when chal-lenged with V. parahaemoly-ticus


? 47

Several strains Artemia culture Decrease of mortality ofArtemia juveniles when chal-lenged with V. proteolyticus

141

a

, not relevant; ?, not specified.



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Fish Eggs and Larvae

There is an urgent need to control the microbiota in hatch-ing incubators by alternative means, since the use of antibioticshas to be minimal. In fact, the use of antibiotics does not allowmicrobial control and may result in an unfavorable alterationof the microbiota. Since it assumed that uncontrolled develop-

ment of the microbial communities in hatcheries is one of themajor reasons for the unpredictable and often variable results,the introduction of microbial control practices by means ofprobiotics may have a beneficial effect on the cultures in hatch-eries. A relatively dense, nonpathogenic, and diverse adherentmicrobiota present on the eggs would probably be an effectivebarrier against colony formation by pathogens on fish eggs (52,84). This rationale has been tried with cod eggs. Hansen andOlafsen (51) attempted to manipulate the egg microbiota ofcod (Gadus morhua) by incubating gnotobiotic eggs in culturesof defined inhibitory bacterial strains; however, these strainsfailed to prevent colonization of the eggs by the microbiotanaturally present in the incubator. Nevertheless, it should benoted that the choice of strains is very important. As discussedbelow, the screening and preselection of potential or putativeprobiotics should be based on extensive experimental workperformed in vivo. Therefore, it would be better if the exper-imental setup described by Hansen and Olafsen (51) was usedas a preselection tool, not to verify the effect of selected bac-teria, as is often done now.

The establishment of a normal gut microbiota may be re-garded as complementary to the establishment of the digestivesystem, and under normal conditions it serves as a barrieragainst invading pathogens. Larvae may ingest substantialamounts of bacteria by grazing on suspended particles and eggdebris (9). It is therefore obvious that the egg microbiota willaffect the primary colonization of the fish larvae.

It has been observed that survival of halibut (Hippoglossushippoglossus) larvae in the first 2 weeks after hatching is af-

fected by incubation with indigenous bacteria isolated fromfish (unpublished data cited in reference 84). Larval survival inthe presence ofVibrio salmonicida-like strains and Lactobacil- lus plantarum amounted to 95%, wheras Vibrio iliopiscariusreduced survival to 63% compared to the control group (81%).

During initial feeding, it is possible to induce an artificialdominance of a certain group of bacteria in the fish-associatedmicrobiota by adding a strain to the rearing water (see, e.g.,reference 123) or to the culture medium of the live food (see,e.g., reference 37). Gatesoupe (37) was able to improve thesurvival rate of larval turbot (Scophthalmus maximus) by dailyaddition of lactic acid bacteria to the enrichment medium ofthe rotifers used as live food for the turbot larvae. The addedlactic acid bacteria could be retrieved in large amounts fromthe turbot larvae, and a significant reduction of larval mortality

was observed when the larvae were challenged with a patho-genic Vibrio on day 9. The hypothesis was that the lactic acidbacteria would act as a microbial barrier against the pathogenicVibrio and might curb the invasion of turbot larvae by thepathogen. Similarly, Garca de la Banda et al. (32) added lacticacid bacteria (Streptococcus lactis and Lactobacillus bulgaricus)to Brachionus and Artemia used in turbot larva feeding. In asingle experiment without replicates, 55% survival was foundon day 17 when living lactic acid bacteria had been added and66% survival was found with disabled ones, as opposed to 34%in the control group. Apparently, the bacterial cells, alive ordisabled, provoked improved survival of the turbot larvae.

Bacillus strain IP5832 spores have been introduced into theculture medium of rotifers, which were fed to turbot larvae(35). A decrease in the proportion of members of the Vibrion-

aceae in the rotifers was observed, and the mean weight of theturbot larvae on day 10 was significantly improved with thespore-fed rotifers. When an experimental infection was per-formed with an opportunistic Vibrio sp., mortality was observedin all treatments but the mean survival of the spore-fed rotiferson day 10 was significantly higher than that of the controlgroup (31 and 10%, respectively). Although the author sug-

gested that the likeliest mode of action is the production ofantibiotics, it is not clear whether an improvement of the nu-tritional status of the larvae could also contribute to the in-creased resistance to infection.

Attention has also been focused on siderophore productionand the probiotic effect of Vibrio type E on turbot larvae (38).The main effect of rotifer enrichment with this strain was toimprove the survival of the larval turbot after a 48-h challengewith the pathogenic Vibrio type P.

Fish Juveniles and Adults

In experiments performed by Gildberg et al. (43), Atlanticsalmon (Salmo salar) fry given a diet supplemented with alactic acid bacterium (related to Lactobacillus plantarum butlater reclassified as Carnobacterium divergens) was challenged with cohabitant fishes infected with Aeromonas salmonicidathrough intraperitoneal injection. Mortality was recorded dur-ing the next 4 weeks. It was shown that lactic acid bacteriagiven as supplements in the dry feed could colonize the intes-tine, but no protection against A. salmonicida infection couldbe detected. Contrary to the expectations, the highest mortalitywas recorded with fish given the diet containing lactic acidbacteria.

Atlantic cod fry fed on dry feed containing lactic acid bac-teria (Carnobacterium divergens) was exposed to a virulentstrain of Vibrio anguillarum. An improved disease resistancewas obtained, and 3 weeks after the challenge lactic acid bac-teria dominated the intestinal microbiota of the surviving fish

given feed supplemented with C. divergens (45). Similarly, twostrains of C. divergens were isolated from the intestine of At-lantic cod and Atlantic salmon and were added to a commer-cial dry feed and administered for 3 weeks to Atlantic cod fry(44). Twelve days after the infection with the same virulent V.anguillarum strain, reduced cumulative mortality was recorded,but 4 weeks after the infection, the same cumulative mortalitywas reached in all groups. Thus, the probiotic treatment onlydelayed the mortality of the cod fry in the infection trials. Theauthors argued, however, that this observation does not ex-clude the considerable importance of such methods under nor-mal rearing conditions in the presence of moderate levels ofopportunistic bacteria.

Jborn et al. (59) studied the ability of Carnobacteriumstrain K1 to colonize the intestinal tract of rainbow trout (On-

corynchus mykiss) (13 to 16 cm long) and to inhibit two com-mon fish pathogens, V. anguillarum and A. salmonicida, inmucus and fecal extracts of rainbow trout. The production ofgrowth inhibitors against both pathogens was demonstrated invitro in both the mucus and the fecal extracts. Furthermore,the Carnobacterium cells remained viable in the intestinal tract,since considerable densities (105 CFU/g) were found in thefecal pellets at least until 4 days after the last feeding. How-ever, a sharp decrease of 3 log units was observed after 3 daysonce the treatment was stopped. Unfortunately, these in vitroobservations have not been confirmed by published in vivodata. Olsson et al. (85) found that the growth of V. anguillarumin fecal extracts from turbot juveniles was inhibited by Car-nobacterium cells. It was concluded that the turbot intestinaltract and feces can serve as an enrichment site for V. anguil-



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larum and that the use of intestinal bacteria with inhibitoryactivity against Vibrio spp. might be used to decrease the loadof fish-pathogenic Vibrio spp. in turbot hatcheries.

Several strains of siderophore-producing Pseudomonas fluo-rescens have been successfully applied as biological controlagents. They were able to exclude a pathogenic A. salmonicidastrain from Atlantic salmon presmolts with stress-inducible

furunculosis infection (117) and to limit the mortality of rain-bow trout (40 g) infected with V. anguillarum (48). Short-termbathing of the fishes in a bacterial suspension of the probiotic(48, 117), long-term exposure in the rearing water (48), or acombination of the two treatments (48) led to a significantdecrease in the mortality after the challenge trial. In bothstudies, a good correlation was found between the productionof siderophores and the protective action of P. fluorescens,suggesting that competition for free iron is involved in themode of action. Smith and Davey (117) concluded that P.fluorescens exerted its effect from the hosts exterior, since thestrain did not significantly invade the fish following bath treat-ment.

A V. alginolyticus strain used as a probiotic in a commercialshrimp hatchery in Ecuador has been applied in a bath treat-ment to Atlantic salmon (21 g) maintained in freshwater (5). V.alginolyticus was found in the intestine up to 21 days after theinitial probiotic application. The challenge experiments re-vealed that the application of the probiont to Atlantic salmonled to a reduction in mortality after exposure to A. salmonicidaand to a lesser extent after exposure to V. anguillarum and V.ordalii.

Crustaceans

Penaeid shrimps. In contrast to the already broad applica-tion of probiotics in commercial penaeid shrimp hatcheries(33, 73; D. R. W. Griffith, Abstr. Larvi95 Fish Shellfish Lar-vicult. Symp. p. 478, 1995); relatively few in-depth studies havebeen published on this subject. Maeda and Liao (68) reported

the use of a soil bacterial strain, PM-4, that promoted thegrowth ofPenaeus monodon nauplii, probably acting as a foodsource. This strain also showed an in vitro inhibitory effectagainst a V. anguillarum strain. When added to tanks inocu-lated with diatoms and rotifers, the strain resulted in 57%survival of the larvae after 13 days, while without the bacteriumall the larvae had died after 5 days (67). Maeda and Liao (69)produced similar data in another study, but the effect wasattributed to strain NS-110.

A V. alginolyticus strain, which was selected based on itsapparent lack of pathogenicity, was inoculated daily into 25-and 60-ton larval rearing tanks containing Litopenaeus van-namei postlarvae (33). The average survival and wet weightwere higher in the tanks containing shrimps that had under-gone bacterial inoculation compared to shrimps receiving pro-

phylactic doses of oxytetracycline and the control group. Tanksof shrimps receiving bacterial inoculation showed no presenceof V. parahaemolyticus in any of the samples taken from theshrimps microbiota, while control tanks and those containingshrimps receiving antibiotics had V. parahaemolyticus in ap-proximately 10% of the samples.

Rengpipat and Rukpratanporn (S. Rengpipat and S. Ruk-pratanporn, Abstr. Fifth Asian Fisheries Forum, p.193, 1998)reported the use of Bacillus strain S11 as a probiotic adminis-tered in enriched Artemia to larvae of the black tiger shrimp(Penaeus monodon). It was found that the P. monodon larvaefed the Bacillus-fortified Artemia had significantly shorter de-velopment times and fewer disease problems than did larvaereared without the Bacillus strain. After being fed for 100 dayswith the Bacillus strain S11-supplemented feed, P. monodon

postlarvae were challenged with a pathogenic V. harveyi strain,D331, by immersion of the shrimps. Ten days later, all thegroups treated with Bacillus strain S11 showed 100% survivalwhereas the control group had only 26% survival (99). Al-though one has to be cautious when comparing the perfor-mance of different aquaculture farms, Moriarty (73) con-cluded, based on his studies in Indonesia, that the use of

several Bacillus cultures in penaeid culture ponds allowed theculture of the shrimps for over 160 days without problemswhereas the farms that did not use the Bacillus cultures expe-rienced almost complete failure in all ponds, with luminescentVibrio disease killing the shrimps before 80 days of culture wasreached. A cost-benefit analysis of the use ofBacillus cultureswas done for a particular farm in Thailand, and under the givencircumstances there was a clear benefit to the producer (74).

Strain BY-9 was produced on large scale and inoculated at106 CFU/ml into an 18-ton larval rearing tank for mass pro-duction of P. monodon larvae. The strain was confirmed toinhibit the in vitro growth ofV. harveyi. The results showed thatBY-9 inoculation gave a lower Vibrio density and a highersurvival rate (46.1 and 10.6%, respectively) than those of thecontrol of larvae cultured up to the tenth postlarval stage(PL-10) (H. K. Sugama and S. Tsumura, Abstr. Fifth AsianFish. Forum, p. 74, 1998).

Crabs. After an inoculation of diatoms and rotifers, thebacterial strain PM-4 was daily introduced for 7 days in 200-m3

tanks containing crab (Portunus trituberculatus) larvae and wasalso inoculated with diatoms and rotifers (67). There was anegative correlation between the presence of PM-4 and thedensities ofVibrio spp. In seven trials, the average survival ofthe crab larvae was 27.2% with strain PM-4. In six of nine trialswithout PM-4, no larvae grew into adults, resulting in an av-erage survival of only 6.8%. These results were reported inthree independent experiments (67, 69, 83).

Bivalve Mollusks

Several studies have focused on the nutritional contributionof probiotics to mollusk larvae; however, no indication wasgiven of their potential biological control abilities (26, 27; J.Torrie and P. Neima, Abstr. Larvi95 Fish Shellfish Larvicult.Symp., p. 489, 1995). A bacterial strain isolated from the go-nads of Chilean scallop ( Argopecten purpuratus) broodstockand characterized as Alteromonas haloplanktis displayed invitro inhibitory activity against the known pathogens V. ordalii,V. parahaemolyticus, V. anguillarum, V. alginolyticus, and Aero-monas hydrophila (106). This A. haloplanktis and a Vibrio strain11 that showed in vitro inhibition of a V. anguillarum-relatedpathogen protected the scallop larvae against the V. anguilla-rum-related pathogen in an experimental infection (105).

Aeromonas media A199 was found to be inhibitory in vitro to

89 strains of aeromonads andVibrio

and could prevent thedeath of oyster (Crassostrea gigas) larvae when they were chal-lenged in vivo with Vibrio tubiashii (42). Administration of theprobiotic strain to the larvae fed with algae caused a spectac-ular decrease of the pathogen densities in the larvae comparedto those in the larvae treated with V. tubiashii only. However,A. media A199 could no longer be detected on the host only 4days after the probiotic treatment, indicating that it would benecessary to introduce the probiotic at regular intervals if aprolonged protective effect is required.

Live Food

Unicellular algae. Unicellular algae are often given as a firstfood or are included in the culture system as a food for rotifersand Artemia. Bacteria increase the growth rate and yield of



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algae (29, 75, 128). However, since bacteria may also inhibitalgal growth (75), careful screening may be necessary whenbacteria are to be used as probiotics in larval rearing or in thegreen-water technique. Rico-Mora et al. (101) selected a strain(SK-05) for its active growth in organic-poor substrates andinoculated it into a Skeletonema costatum culture. When allcultures had reached the late exponential phase, Vibrio algino-

lyticus was inoculated as a typical contaminant. After 48 h itwas shown that strain SK-05 prevented the proliferation of V.alginolyticus, although it exerted no in vitro inhibitory actionagainst V. alginolyticus. It was suggested that the protectiveeffect was due to competitive exclusion, since only strain SK-05was able to utilize the exudates ofS. costatum.

Rotifers. Several studies have been published on the nutri-tional contribution of lactic acid bacteria (see e.g., references34, 36, 39, and 55) and other bacteria (see e.g., references 50,and 108; P. Bogaert, M. Dehasque, and P. Sorgeloos, Abstr.World Aquacult. 93, p. 186, 1993) to the production rate of therotifer Brachionus plicatilis. The control of the microbiota ofrotifer cultures has received less attention. Under optimal cul-ture conditions, no increase of the growth rate of the rotiferswas observed after addition of Lactococcus lactis AR21 to thediet. Under a suboptimal feeding regime where the amount offood was reduced by 55%, L. lactis AR21 counteracted thegrowth inhibition of the rotifers due to V. anguillarum in two ofthe three experiments performed. It was not possible, however,to recover either L. lactis AR21 or V. anguillarum from therotifers after 24 h (54). Live food such as rotifers is oftensuspected of being a vector for bacterial infections of the pred-atory organisms (76, 81, 89, 129). It is therefore surprising thatstudies dealing with the proliferation of larval pathogens inrotifer cultures are so scarce. Only Gatesoupe (36) reportedthat the proliferation of Aeromonas salmonicida, accidentallyappearing in the experimental rotifer culture, was inhibited bythe presence of Lactococcus plantarum as a supplement.

Artemia. The capacity of V. alginolyticus C14 to prevent

mortality in Artemia nauplii was demonstrated by Gomez-Gilet al. (47); i.e., inoculation of strain C14 before the challengeprevented Artemia nauplii from dying after being experimen-tally infected with V. parahaemolyticus HL58, although strainHL58 was still able to colonize the nauplii (47).

Verschuere et al. (140) selected nine bacterial strains thatpositively influenced the growth and/or survival of juveniles ofthe brine shrimp Artemia cultured as a live food for otherspecies. All nine strains were able to delay the death of theArtemia when experimentally infected with the pathogenic V.proteolyticus CW8T2, although large differences were foundamong the strains. While all Artemia in the axenic control died within 2 days after the infection, the survival rates of theArtemia cultures inoculated beforehand with strain LVS8 or amixture of the nine strains showed more than 80% survival

after 4 days (141). Furthermore, the growth ofV. proteolyticusCW8T2 in the Artemia culture water was considerably slowedin the presence of strain LVS8.

Microbially Matured Water

Although it is not strictly a probiotic treatment, attemptshave been made to optimize the rearing water for larvae ofseveral marine species by so-called microbial maturation (115,135). Microbial maturation of seawater prepared by transientmaintenance in a maturation tank with a biofilter led to asignificantly higher initial growth rate of turbot larvae than inmembrane-filtered water. Proliferation of opportunistic bacte-ria was observed in the rearing water after hatching of theturbot eggs, but it occurred to a lesser extent in the microbially

matured water (115). Also, clear differences in survival of hal-ibut yolk sac larvae were observed (135). The experimentssupported the hypothesis that microbial maturation selects fornonopportunistic bacteria that protect the marine larvae fromthe proliferation of detrimental opportunistic bacteria (115).

Interaction with Nutritional Effects

As mentioned above, it is sometimes impossible to separatefeeding aquatic organisms from environmental control. Addi-tion of bacteria to the rearing water of filter feeders such asrotifers, bivalve larvae or adults, and crustacean larvae mayresult in massive uptake of these bacteria, possibly acting as a(complementary) food source or contributing to the digestionof the food, even if the main goal of the probiotic applicationwas, e.g., suppression of a pathogen in the culture water. It issometimes unclear whether the probiotic effect is attributed tosuppression of a pathogen or if it is a direct or indirect conse-quence of the nutritional effect of the probiotic.

This duality has been addressed by Riquelme et al. (105) andVerschuere et al. (140, 141). Riquelme et al. (105) evaluatedthe effect of 11 bacterial strains that were inhibitory in vitro

toward a V. anguillarum-related pathogen on the survival ofChilean scallop larvae when they were fed only the differentinhibitory bacterial strains for 14 days. Only two strains (Vibriostrains 11 and 334) had no detectable effects on the scalloplarvae, while the other strains resulted in high mortality. In anin vivo challenge test, only Vibrio strain 11 had a protectiveeffect against infection with the V. anguillarum-related patho-gen. Verschuere et al. (140, 141) inverted the approach andselected nine bacterial strains that enhanced to the nutritionalvalue of the dry food for Artemia juveniles. The in vivo antag-onism of the nine strains for the pathogen V. proteolyticusCW8T2 was examined in experimental-infection trials, andthey showed various degrees of protection. These results indi-cate that the two aspects must be examined separately, but it is

conceivable that a combination of nutritional contribution anddisease control yields the best probiotic effect.

POSSIBLE MODES OF ACTION

Although many publications about probiotics in aquaculturehave emerged during the last decade, the approach was gen-erally empirical and the arguments with regard to the mode ofaction were often circumstantial. The exact modes of action ofthe probiotics were rarely completely elucidated. In humanand agriculture application, probiotic research has enjoyedmuch more attention through history and several modes ofaction have been supported by unambiguous experimentaldata (see the review by Fuller [30]). It is clear that the expe-rience obtained with terrestrial animals has been used in

aquaculture, especially with regard to the use of lactic acidbacteria. Also, the suppression of plant root pathogens byfluorescent Pseudomonas spp. has been examined in detail (seethe review by OSullivan and OGara [88]). Although each ofthese disciplines has its specific characteristics, several of themicrobial-ecological mechanisms involved are similar, allowingsome transfer of experimental evidence.

Considering the possible probiotic effect in vivo, one has tomake a distinction between the intrinsic ability of the strain topositively influence the host and its ability to reach and main-tain itself in the location where the effect is to be exerted. Forinstance, the production of siderophores or inhibitory com-pounds in sufficient amounts and even under the conditionsprevailing in the gut is of no relevance if the strain is notingested by the host. This is important, since Prieur (93) has



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demonstrated both selective ingestion and digestion of mi-crobes by the bivalve Mytilus edulis. Similarly, if the candidateprobiotic is not capable of efficient proliferation in the gut afterbeing ingested, it is improbable that it will exert strong effectsunless it is added regularly through the diet. Hence, the pos-sible modes of action require implicitely that the candidateprobiotics be able to reach the location where their probiotic

effect is required. Those modes are as follows: production ofinhibitory compounds; competition for chemicals or availableenergy; competition for adhesion sites; enhancement of theimmune response; improvement of water quality; interactionwith phytoplankton; source of macro- and micronutrients; andenzymatic contribution to digestion. The last two of these arenot dealt with in this review. The possible modes of action arediscussed in more detail in the following sections.

Production of Inhibitory Compounds

General aspects. Microbial populations may release chemi-cal substances that have a bactericidal or bacteriostatic effecton other microbial populations, which can alter interpopula-tion relationships by influencing the outcome of competitionfor chemicals or available energy (28, 62, 94). The presence ofbacteria producing inhibitory substances in the intestine of thehost, on its surface, or in its culture medium is thought toconstitute a barrier against the proliferation of (opportunistic)pathogens.

In general, the antibacterial effect of bacteria is due to thefollowing factors, either singly or in combination: productionof antibiotics (145), bacteriocins (15, 94, 136), siderophores(which are dealt with in a later section), lysozymes, proteases,and/or hydrogen peroxide and the alteration of pH values bythe production of organic acids (126). Vandenbergh (136)added the formation of ammonia and diacetyl to this list.

Lactic acid bacteria are known to produce compounds suchas bacteriocins that inhibit the growth of other microorganisms

(136). There are many reports of the inhibitory activity of lacticacid bacteria mediated by bacteriocins, mostly but not exclu-sively against gram-positive bacteria (reviewed by Piard andDesmazeaud [91]) (121). However, almost all the pathogensinvolved in aquaculture are gram negative. The possible in-volvement of lactic acid bacteria as probiotics in aquaculture isdiscussed by Ring and Gatesoupe (103). Since lactic acidbacteria normally account for only a small part of the gutmicrobiota of fish and since they are generally considered to benonpathogenic (with some exceptions), it can be questioned ifthe inhibition of closely related species by bacteriocins pro-duced by lactic acid bacteria can effectively contribute to thehealth status of the higher organism.

Compounds other than bacteriocins and antibiotics havebeen suggested to play a role in the amensalism that may occur

between bacterial species. Nair et al. (79) showed that a largeproportion of marine bacteria produced bacteriolytic enzymesagainst V. parahaemolyticus. Furthermore, Imada et al. (57, 58)isolated and characterized Alteromonas sp. strain B-10-31, iso-lated from nearshore seawater of Japan, which produces analkaline protease inhibitor called monastatin. In an in vitroassay, the purified and concentrated monastatin showed inhib-itory activity against a protease from Aeromonas hydrophilaand a thiol protease from V. anguillarum, both pathogenic tofish.

Many authors assign the inhibitory effects detected in in vitro antagonism tests to bacteriocins or antibiotics withoutlooking for any other causes. It has been argued that observedgrowth inhibition can, in many cases, be accounted for byprimary metabolites or simply by a decrease of the pH (6, 131).

Unless the inhibitory compounds have been identified, in thisreview we use the expression inhibitory compounds ratherthan antibiotics or bacteriocins.

Production in aquaculture. Many studies have demon-strated the presence of bacterial strains showing in vitro inhi-bition toward pathogens known to occur in aquaculture (3, 5,6, 24, 4345, 50, 54, 59, 63, 83, 86, 99, 105, 110, 126, 127, 130,

143). This shows that the ability to inhibit other bacteria is notuncommon for bacteria found in aquaculture environments.However, it has not been demonstrated that production ofsuch inhibitory compounds occurs under in vivo conditions,and the ecological relevance of the production of inhibitorycompounds toward other bacteria is still unclear.

The in vitro production of inhibitory compounds towardknown pathogens for the considered species has often beenused in the selection of putative probiotic strains (see, e.g.,references 42, 51, 99, and 105). At this stage, however, theassociation between amensalistic activity and in vivo probioticactivity is very weak and circumstantial. Typically, a correlationis made between the in vitro ability of the probiotics to inhibitpathogens and the in vivo protection of the cultured aquaticspecies, but in none of the studies published so far has it beenshown unequivocally that the production of inhibitory com-pounds is the cause of the observed in vivo probiotic activity ofthe strains. Hence, future research in this field is required.Similar research has already been performed in the field ofplant disease suppression, where the production of inhibitorycompounds by some fluorescent Pseudomonas spp. is now rec-ognized as an important factor in the disease suppression abil-ity of these strains (see the review by OSullivan and OGara[88]).

Competition for Chemicals or Available Energy

General aspects. Competition for chemicals or available en-ergy may determine how different microbial populations coex-

ist in the same ecosystem (28). On theoretical grounds, itseems likely that competition for nutrients will operate in themammalian gut, but the evidence for its occurrence in humansand terrestrial animals is not good (30). Competition for nu-trients can theoretically play an important role in the compo-sition of the microbiota of the intestinal tract or ambient en-vironment of cultured aquatic species, but to date there havebeen no comprehensive studies on this subject (103). Hence,successful application of the principles of competition to nat-ural situations is not easy and remains a major task for micro-bial ecologists.

The microbial ecosystem in aquaculture environments isgenerally dominated by heterotrophs competing for organicsubstrates as both carbon and energy sources. Specific knowl-edge of the factors governing the composition of the micro-

biota in aquaculture systems is required to manipulate it. Thisknowledge, however, is generally not available, and one musttherefore rely on an empirical approach. Nevertheless, Rico-Mora et al. (101) selected a bacterial strain for its active growthin organic-poor substrates and inoculated it into a diatomculture, where it prevented the establishment of an introducedV. alginolyticus strain. Since the inoculated strain had no invitro inhibitory effect on V. alginolyticus, it was suggested thatthe strain was able to outcompete V. alginolyticus due to itsability to utilize the exudates of the diatom.

Verschuere et al. (140) selected several strains with a posi-tive effect on the survival and growth ofArtemia juveniles. Thein vitro antagonism tests and filtrate experiments showed thatno extracellular inhibitory compounds were involved in theprotective action of these strains against V. proteolytics CW8T2



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but that living cells were required to protect Artemia againstthe pathogen. It was suggested that the selected bacteria ex-erted their protective action by competing with the pathogenfor chemicals and available energy (141).

Competition for iron. Virtually all microorganisms requireiron for growth (98). Siderophores are low-molecular-weight(1,500), ferric ion-specific chelating agents (80) which can

dissolve precipitated iron and make it available for microbialgrowth. The ecological significance of siderophores resides intheir capacity to scavenge an essential nutrient from the envi-ronment and deprive competitors of it. Successful bacterialpathogens are able to compete successfully for iron in thehighly iron-stressed environment of the tissues and body fluidsof the host (147). The ecological significance of siderophores insoils as important tools for iron acquisition by microorganismsand plants and their involvement in suppression of plant rootpathogens have been established (12, 88).

The omission of iron from the diet of early-weaned seabass(Dicentrarchus labrax) larvae had no detrimental effect on thesurvival or the growth rate of the fish but significantly limitedthe bacterial load of the larvae and increased the diversity ofthe microbiota (40). The requirement for iron is high for manypathogens, including V. anguillarum. In a challenge test withthis bacterium, salmon mortality increased linearly with dietaryiron content (40, 109). Both observations indicate the impor-tance and the biological role of ferric iron in the establishmentof the microbiota associated with cultured aquatic species.

Harmless bacteria which can produce siderophores could beused as probiotics to compete with pathogens whose pathoge-nicity is known to be due to siderophore production and com-petition for iron or to outcompete all kind of organisms re-quiring ferric iron from solution. The possible effectiveness ofsiderophore-producing probiotics can be illustrated by thestudy of Gatesoupe (38), in which the addition of the bacterialsiderophore deferoxamine to live food (rotifers) increased theresistance of turbot larvae challenged with the pathogenic

Vibrio strain P. The addition of a siderophore-producing Vibriostrain E protected the turbot larvae slightly more.Pybus et al. (94) tested thirty strains of V. anguillarum as

potential probiotics against the salmon pathogen V. ordalii bythe deferred-antagonism test. Only one strain (V. anguillarumVL4335) inhibited strains of V. ordalii in vitro, and this effectwas blocked when iron salts were added to the medium, indi-cating that the growth inhibition was linked to iron deficiency.Using the chrome-azurol sulfate assay to measure siderophoreproduction, V. anguillarum VL4335 yielded significantly highervalues than other V. anguillarum strains.

Smith and Davey (117) showed that the fluorescent pseudo-monad F19/3 is capable of inhibiting the growth of Aeromonassalmonicida in culture media and that this inhibition is due tocompetition for free iron. Strain F19/3 was also capable of

excluding A. salmonicida from Atlantic salmon (Salmo salar)presmolts with stress-inducible infections.

Gram et al. (48) examined the applicability of the sid-erophore-producing Pseudomonas fluorescens AH2 isolatedfrom frozen freshwater fish. This latter strain is inhibitory toseveral gram-positive and gram-negative bacteria, particularlywhen iron availability is limited. In vitro tests revealed that thegrowth of V. anguillarum was inhibited by the filter-sterilizedsupernatans from iron-limited cultures of P. fluorescens AH2but not from iron-replete cultures. During coculture, P. fluo-rescens AH2 inhibited the growth of V. anguillarum indepen-dently of the iron concentration, when the initial level of thesiderophore producer was 100 to 1000 times greater than thelevel of the fish pathogen. The in vitro tests were confirmed byan in vivo antagonism test, in which the mortality of rainbow

trout juveniles due to V. anguillarum infection was decreasedby 46% when the culture was treated with P. fluorescens AH2.

Siderophore production by fluorescent pseudomonads insoil is known to be influenced by a great variety of factors (12,88). Therefore, detection of in vitro production of sid-erophores does not necessarily mean that they are produced insignificant amounts in vivo in order to have a significant bio-

logical control effect. Similar to the production of inhibitorycompounds, the evidence for the participation of competitionfor chemicals or available energy and, more specifically, of freeiron or siderophores in the mode of action of probiotics is stillcircumstantial.

Competition for Adhesion Sites

One possible mechanism for preventing colonization bypathogens is competition for adhesion sites on gut or othertissue surfaces. It is known that the ability to adhere to entericmucus and wall surfaces is necessary for bacteria to becomeestablished in fish intestines (86, 87, 143). Since bacterial ad-hesion to tissue surface is important during the initial stages ofpathogenic infection (61), competition for adhesion receptors

with pathogens might be the first probiotic effect (72). In hu-man medicine, adherent strains are key candidates for probi-otic therapy (113).

Adhesion can be nonspecific, based on physicochemical fac-tors, or specific, involving adhesin molecules on the surface ofadherent bacteria and receptor molecules on epithelial cells(113). Inhibition of the adhesion of pathogens to human orother mammal cells has already been demonstrated in vitro byseveral authors (8, 10, 19, 21). Competitive exclusion resultingfrom preemptive colonization has been shown for the cecalwalls of chickens, which kept their effect after the ceca werewashed four times in buffered saline (120). As far as is known,similar research approaches have not yet been followed withaquatic species, and competition for adhesion sites as a modeof probiotic action is still hypothetical.

Adhesion capacity and growth on or in intestinal or externalmucus has been demonstrated in vitro for fish pathogens likeV. anguillarum and A. hydrophila (31, 61) and for candidateprobiotics such as Carnobacterium strain K1 (59) and severalisolates inhibitory to V. anguillarum (86). In one of these stud-ies, the aim was to measure the in vitro capacity of the strainsto adhere to and grow in turbot intestinal mucus in order toinvestigate their potential to colonize farmed turbot as a meansof protecting the host from infection by V. anguillarum (86).The intestinal isolates generally adhered much better to a filmof turbot intestinal mucus, skin mucus, and bovine serum al-bumin than did V. anguillarum, indicating that they could com-pete effectively with the pathogen for adhesion sites on themucosal intestinal surface.

Adhesion of probiotics to the gut wall or other tissues doesnot necessarily imply competition for adhesion sites as the(only) mode of action for the probiotic effect. It is conceivablethat bacteria are able to colonize, for example, the intestinalgut wall of a fish and exert their protective action against apathogen by excreting inhibitory compounds.

Enhancement of the Immune Response

Immunostimulants are chemical compounds that activatethe immune systems of animals and render them more resis-tant to infections by viruses, bacteria, fungi, and parasites (96).Fish larvae, shrimps, and other invertebrates have immunesystems that are less well developed than adult fish and aredependent primarily on nonspecific immune responses fortheir resistance to infection (118).



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Observations obtained in experiments with warm-bloodedanimals indicate that probiotic (lactic acid) bacteria adminis-tered orally may induce increased resistance to enteric infec-tions (56). There are many reports that bacterial compoundsact as an immunostimulant in fish and shrimp, as has beenreviewed recently (111), but only specific cell compounds ornonliving cells were used in these studies. It has also been

suggested that ingestion of bacteria and subsequent endocyto-sis in cod and herring larvae are involved in stimulation of thedeveloping immune system (84). However, at present it is notclear whether bacteria administrered as probiotics could havea beneficial effect on the immune response of cultured aquaticspecies, but such a mode of action cannot a priori be excluded.

Improvement of Water Quality

In several studies, water quality has been recorded duringthe addition of the probiotics, especially Bacillus spp. Therationale is that gram-positive Bacillus spp. are generally moreefficient in converting organic matter back to CO

2than are

gram-negative bacteria, which would convert a greater per-centage of organic carbon to bacterial biomass or slime (119).

It is reasoned that by maintaining higher levels of these gram-positive bacteria in the production pond, farmers can minimizethe buildup of dissolved and particulate organic carbon duringthe culture cycle while promoting more stable phytoplanktonblooms through the increased production of CO

2(114). How-

ever, several studies utilizing one or more bacterial speciessuch as Bacillus, Nitrobacter, Pseudomonas, Enterobacter, Cell-ulomonas, and Rhodopseudomonas spp. in the culture ofshrimps (65, 99) or channel catfish (13, 20, 95, 133) could notconfirm this hypothesis. This shows that the published evidencefor water quality improvement is poor, except with regard tonitrification.

The start-up of biofilters by transferring medium from anexisting filter is a common practice in aquaculture. However,the use of nitrifying cultures as inocula to speed up the estab-

lishment of nitrification is not so common. A lot of bacterialcultures containing nitrifying bacteria to control the ammonialevel in culture water are available commercially and are aimedespecially at aquarium hobbyists. Nitrifiers are responsible forthe oxidation of ammonia to nitrite and subsequently to ni-trate. Seeding of biological filters with nitrifying bacteria iseffective in reducing the activation time of new biofilters (18).Perfettini and Bianchi (90) used inocula consisting of frozencells to accelerate the conditioning of new closed seawaterculture systems, and the time to establish nitrification wasshortened by about 30%. In a commercial semiclosed waterrecirculation system, the start-up of a nitrifying biofiter wasreduced from 34 weeks to 10 days by inoculation of a veryactive liquid culture of nitrifiers (I. Van Hauteghen, G. Rom-

baut, and W. Verstraete, Abstr. Int. Grf. AQUA 2000, p. 338,2000).Nitrifying cultures could also be added to the ponds or the

tanks when an incidental increase of ammonia or nitrite levelsis observed. Besides ammonia, nitrite toxicity is a commonproblem in fish culture (64), for example in pond rearing ofcatfish (134).

Interaction with Phytoplankton

Recent reports demonstrate that many bacterial strains mayhave a significant algicidal effect on many species of microal-gae, particularly of red tide plankton (29). Of 41 bacterialstrains tested, 23 inhibited the growth of the unicellular alga Pavlova lutheri to various degrees (75). Bacteria antagonistictoward algae would be undesirable in larval rearing where

unicellular algae are added (e.g., the green-water technique)but would be advantageous when undesired algal species de-velop in the culture pond.

Claims for bacterial amendments include a decrease of theproportion of cyanobacteria. One such bacterial suspensionconsisted of Bacillus, Nitrobacter, Pseudomonas, Enterobacter,Cellulomonas, and Rhodopseudomonas spp. and was applied to

channel catfish ponds (13). However, the percentages of cya-nobacteria did not differ between the treatment and the con-trol on any of the sampling dates.

Positive effects of bacteria on cultured microalgae have alsobeen observed (29, 75, 101, 128) (see above). It is conceivablethat bacteria can indirectly influence the health or the zootech-nical performance of the cultured aquatic animals throughtheir effect on the microalgae used as food or in the green-water technique. When probiotic bacteria are selected to beused in a culture environment comprising algae, their possibleinteraction with these unicellular algae must be taken intoconsideration when the mode of action is being investigated.

RATIONALE FOR SELECTING AND DEVELOPING

PROBIOTICS IN AQUACULTUREThe development of probiotics applicable to commercial use

in aquaculture is a multistep and multidisciplinary process re-quiring both empirical and fundamental research, full-scaletrials, and an economic assessment of its use. This sectiondiscusses the different crucial phases which must be covered inorder to develop effective and safe probiotics. In Fig. 1 thisrationale is presented schematically.

Acquisition of Background Information

Before research and development activities are begun, bot-tlenecks in culture practices or in economic development ofthe aquaculture farm or industry should be identified, takinginto account possible restricting or directing legislative deci-

sions. A critical review of the available scientific literature anda profound knowledge of the rearing practices used in aqua-culture farms should determine whether a probiotic approachwould be feasible and is worth further examination. The abi-otic and biotic environment inhabited by the cultured organ-isms should be well characterized, with particular attentionbeing paid to the microbiota, the relationships between themicrobiota (pathogens and others) and the host, and the rela-tionships between the microbiota and the carrying capacity ofthe culture environment.

Acquisition of Putative Probiotics

The acquisition of a good pool of candidate probiotics is ofmajor importance in this process. It is vital in this phase that

the choice of strains is made as a function of the possible roleof the probiotics to be developed, although there is no un-equivocal indication that putative probiotics isolated from thehost or from their ambient environment perform better thanisolates completely alien to the cultured species or originatingfrom a very different habitat (Table 1). There are several re-ports of the use in aquaculture of probiotics developed forhumans or terrestrial animals (34, 35, 37; Bogaert et al., Abstr.World Aquacult. 93). However, there is an elegant logic inisolating putative probiotics from the host or the environmentin which the bacteria are supposed to exert their probioticeffect. It is assumed that strains showing a dominant coloniza-tion of, e.g., the intestinal mucus of fish are good candidates tocompetitively exclude pathogens from the adhesion sites of thegut wall. Similarly, the presence of a dominant bacterial strain



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in high densities in culture water indicates its ability to growsuccessfully under the prevailing conditions, and one can ex-pect that this strain will compete efficiently for nutrients withpossibly deleterious strains. Identification of the isolates at thisstage is not essential.

Screening and Preselection of Putative Probiotics

Generally at the end of the previous phase, one ends up witha pool of isolates that must be screened and preselected toobtain a restricted number of isolates for further examination.Several approaches followed in literature are discussed below.The application of in vitro tests to screen the acquired bacterialstrains presupposes a well-known mode of action to select theappropriate in vitro test. Since the evidence about the possible

modes of action of probiotics is still equivocal, preferenceshould be given to in vivo tests in the search for probiotics. Theuse of the target organism in the screening procedure providesa stronger basis.

In vitro antagonism tests. A common way to screen thecandidate probiotics is to perform in vitro antagonism tests, in which pathogens are exposed to the candidate probiotics ortheir extracellular products in liquid (43, 45, 48) or solid (2, 4,6, 24, 42, 48, 63, 71, 83, 86, 105, 126, 127, 143) medium.Depending on the exact arrangement of the tests, candidateprobiotics can be selected based on the production of inhibi-tory compounds (43, 48, 59, 63, 86, 117) or siderophores, or onthe competition for nutrients (24).

The results of in vitro antagonism tests should be interpretedwith great caution. Olsson et al. (86) concluded that growth in

FIG. 1. Rationale for the research and development of probiotics as biological control agents in aquaculture.



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marine broth or on marine agar produced fewer detectableinhibitory metabolites than did growth in tryptic soy broth.This is in agreement with the findings of Mayer-Harting et al.(71), who noted that the composition of the medium mightaffect the amount of bacteriocin produced or the amount re-leased into the medium. In an attempt to overcome this prob-lem, Jborn et al. (59) performed in vitro antagonism tests

between Carnobacterium strain K1 and pathogenic V. anguil-larum and A. salmonicida in intestinal mucus and fecal extractsof rainbow trout. Furthermore, growth inhibition may not al- ways be a consequence of the production of inhibitory sub-stances like antibiotics, but inhibition caused by primary me-tabolites or changes in pH is also possible (6, 131).

The preselection of candidate probionts based on these invitro antagonism tests has often led to the finding of effectiveprobiotics (see, e.g., reference 42). It is unclear, however,whether the finding of a good probiotic and the criterion forthe preselection are connected. In other words, it has not beenshown that the isolates that did not inhibit the pathogens in thein vitro antagonism test were less effective in protecting thehost against pathogenic effects in vivo. It has not been dem-onstrated unequivocally that there is a representative in vitroassay which can provide an extra criterion in the selectionprocess. According to Ring and Gatesoupe (103), neither apositive nor a negative in vitro test may predict the actual effectin vivo.

Colonization and adhesion. It is stated above that a candi-date probiotic should either be supplied on a regular basis orbe able to colonize and persist in the host or in its ambientenvironment. The ability of a strain to colonize the gut or anexternal surface of the host and adhere to the mucus layer maybe a good criterion for preselection among the putative pro-biotics. This involves the viability of the potential probioticwithin the host and/or within its culture environment, adher-ence to host surfaces, and the ability to prevent the establish-ment of potentially pathogenic bacteria. Examination of adhe-

sion properties using intestinal cells has become a standardprocedure for selecting new probiotic strains for human appli-cation (113), but it is less common in aquaculture (59, 86).

Small-scale tests, with particular attention to monoxeniccultures. (i) Small-scale tests (xenic). Sea scallop (Placopectenmagellanicus) larvae, 6 to 8 days old, were cultured in thepresence of several bacterial isolates. After 4 days, the healthof the larvae was measured by comparing their sizes, swimmingpatterns, and feeding behaviors. From this, bacteria were iden-tified as neutral, potential pathogens, or possible probionts(Torrie and Neima, Abstr. Larvi 95). The rearing of marinefish and mollusk larvae on a small scale allows the screening ofmany candidate probiotics in the presence of the target organ-ism. Small-scale in vivo antagonism tests were performed inplastic multiwell dishes during the yolk sack stage of turbot and

halibut larvae, allowing representative and reproducible exam-ination of putative probiotics (. Bergh and L. Torkildsen,Abstr. Eighth Int. Symp. Microb. Ecol., p. 104, 1998).

(ii) Monoxenic cultures. To determine the effects of a spe-cific bacterial strain on a cultured organism, the elimination ofother microbes from the culture system may be necessary toavoid microbial interactions (16). For example, several authorshave reported that the nutritional value of the food ofArtemiadepends partly on the spontaneous colonization of the foodparticles by harmless bacteria (22, 25).

A first selection of candidate probiotics can be performed byculturing the fish species under monoxenic conditions, i.e., onlyin the presence of the putative probiotic as a bacterial species.This approach has already been used to study the effects ofseveral bacterial strains on cultures of unicellular algae (29, 75,

128), the rotifer Brachionus plicatilis (82, 108), the brine shrimpArtemia (140), turbot larvae (75, 82), and larvae of the oysterCrassostrea gigas (27, 82). Generally these cultures are per-formed on a small scale, where all inputs (culture water, feed,etc.) are sterile and where the eggs, larvae, or unicellular algaeare harvested under sterile conditions or disinfected before theexperiment. The limiting factor in this approach, however, is

the availability of large numbers of axenic organisms. There-fore, this approach has been used only with live food organisms(unicellular algae, rotifers, and Artemia) or larvae of mollusksor fishes.

Evaluation of Pathogenicity of Selected Strains

Before a culture can be used as a probiotic, it is necessary toconfirm that no pathogenic effects can occur in the host. There-fore, the target species should be challenged with the candi-date probiotic, under normal or stress conditions. This can bedone by injection challenges, by bathing the host in a suspen-sion of the candidate probiotic, or by adding the probiotic tothe culture. Garriques and Arevalo (33) examined the patho-genicity of three bacterial isolates toward Litopenaeus van-namei by adding these to the nauplii cultures. Mortality wasrecorded for up to 4 days, and the animals were monitored forphototactic response. Austin et al. (5) injected 21-g Atlanticsalmon intramuscularly or intraperitoneally with a suspensioncontaining the candidate probiotic. The fish were monitoredfor 7 days, after which the survivors were sacrificed and exam-ined for disease symptoms by examining the kidneys, spleenand muscles.

This phase in the search for probiotics can be combined withthe previous one when small-scale in vivo tests are performed,but it should preferably be done under monoxenic conditionsto eliminate every interaction with the already established mi-crobiota. Other trophic levels involved in the culture of thetarget organism should also be taken into account in the re-

search and development process. When probiotics are beingdeveloped to biologically control and/or increase live-food pro-duction, pathogenicity toward the predator larvae or juvenilesshould also be considered. To test this, Verschuere et al. (L.Verschuere, G. Rombaut, R. Robles, Y. Yaodong, P. Sorge-loos, and W. Verstraete, Abstr. Eighth Int. Symp. Microb.Ecol., p. 338, 1998) injected 125-day-old juveniles of Litope- naeus vannamei with a mixture of nine probiotic strains forArtemia juveniles. One day after injection, survival was mea-sured and samples of hemolymph were plated on marine agarto see whether the defense systems of the shrimps were able tocope with the intrusion. Similarly, unicellular algae may beinhibited or even stimulated by bacterial isolates from, e.g.,turbot larvae (75). When probiotics are selected for larvalrearing by the green-water technique, their possible interaction

with algae should be considered.

In Vivo Evaluation of Potential Probiotic Effects on the Host

The effect of candidate probiotics should be tested in vivo aswell. When the probiotic effect is supposed to be nutritional,the candidate probiotics could be added to the culture of theaquatic species and their effect on growth and/or survival pa-rameters could be assessed. However, when biological controlof the microbiota is desired, representative in vivo challengetests seem to be the appropriate tool to evaluate the potentialeffect of the candidate probiotic on the host.

Mode of application of the putative probiotic. The putativeprobiotic can be added to the host or its ambient environmentin several ways: (i) addition to the artificial diet, (ii) addition to



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the culture water (140), (iii) bathing (5, 117), and (iv) additionvia live food (23, 46, 107).

Experimental infectionsin vivo antagonism tests. Thenext important step in the in vivo challenge test involves ex-perimental infection with a representative pathogen. Patho-gens or opportunistic pathogens can be administered via thediet (live food or artificial), through immersion, or via the

culture water, similarly to the probiotics. In experiments per-formed by Gildberg et al. (43), Atlantic salmon fry was chal-lenged with cohabitant fishes which were previously infectedwith Aeromonas salmonicida through intraperitoneal injection.The mortality of the challenged fishes due to acute furunculo-sis was monitored over time.

In vivo challenge tests to detect probiotic effects should beperformed with great caution. The long-term effects should bestudied to determine how the pathogen is evolving. It is im-portant to observe whether the growth or the activity of thepathogen in the location where the antagonism of the probioticis expected is really suppressed or if it is only delayed, maybesimply due to some competition for nutrients. This can beillustrated by the work of Gildberg and Mikkelsen (44). Attwelve days after infection of Atlantic cod fry with a pathogenicV. anguillarum strain, reduced cumulative mortality was ob-served in fish given feed supplemented with Chaetoceros diver-gens. At 4 weeks after the infection, however, the same cumu-lative mortality as in the control treatment was reached. Theauthors argued that this does not exclude the possibilitythough, that such methods may be of considerable importanceunder normal rearing conditions in the presence of moderatelevels of opportunistic bacteria. In contrast, Gibson et al. (42)observed a clear decrease in the level of the pathogen V.tubiashii in a culture of oysters when Aeromonas media wasadded, although the probiotic strain itself could no longer bedetected in the culture after 4 days. This shows the necessity torecord mortality or disease over a long period after experimen-tal infection. Moreover, the pathogen should be quantified

regularly in the culture.

Mass Production, Economic Evaluation, andEvaluation of Compliance with legislation

The approach outlined above should result in a set of strainswith a well-established probiotic effect on the target organism without affecting other possibly involved trophic levels. Com-parative pilot experiments under hatchery or growout con-ditions in the farms should be performed to estimate theeconomic consequences of the probiotic application. An im-portant factor in the economic evaluation is the mass produc-tion of the probiont. Also, effective legislation, if any, should betaken into account before commercial application is begun.Finally, a cost-benefit analysis will determine whether the pro-

biotics could be applied in practice or not.

Development of Monitoring Tools

Once it is decided to apply probiotics on a large scale, it isnecessary to develop monitoring tools to control the produc-tion and application of the bacterial cultures. Production oflarge amounts of bacterial biomass requires appropriate qual-ity control to avoid contamination by other bacteria. Attentionshould be paid to the genetic stability of the produced bacterialbiomass, since spontaneous or induced mutations could affectthe probiotic activity of the bacterial strain(s). Monitoringshould also be performed following the administration of theprobiotic cultures to the cultured aquatic species. Monitoringthe microbiota in the culture system should become part of the

routine practice in hatcheries. Molecular tools may be mostappropriate for this kind of application.

CONCLUSIONS AND FURTHER DIRECTIONS

This review shows that experimental evidence is accumulat-ing that the health and zootechnical performances of cultured

aquatic species can be improved by the prophylactic use ofprobiotics. Progress has been made in the culture of live food,crustacea, mollusks, and fish. The use of probiotics as biolog-ical control agents should be considered to be a kind of riskinsurance that may not provide any notable benefit when theculture is performing under optimal conditions and in theabsence of (opportunistic) pathogens but that will be veryhelpful if infectious diseases break out.

The scarcity of data about their effective implementation inpractice makes the practical relevance of probiotics in aqua-culture hazy as yet. Furthermore, there is still a lack of knowl-edge about the exact modes of action involved in probioticeffects. Typically, a correlation is made between, for instance,the in vitro observations that a pathogen is inhibited by theproduction of an antimicrobial or bacteriostatic agent or bycompetition for nutrients (e.g., production of a siderophore)and the in vivo probiotic effect of the same bacterial strain. Itcan be argued that it is unlikely from an evolutionary point ofview that bacteria spend a great deal of energy and resourcesif this effort is not beneficial to the organism in one way oranother, but the evidence is still circumstantial and not con-clusive. More in-depth studies of the competitive processesbetween bacteria must be carried out, and the ecological rel-evance of the different processes in situ remains to be eluci-dated. Also, the interaction between the cultured aquatic spe-cies and its associated microbiota deserves further researchinto possible immunostimulative effects.

Most of the literature references on the use of probioticsreport on probiotics consisting of single bacterial strains (Table

1). It stands to reason that probiotics based on a single strainare less effective than mixed-culture probiotics when microbialcontrol is desired. The approach should be systemic, i.e., basedon a mixture of versatile strains capable of acting and inter-acting under a variety of conditions and able to maintain them-selves in a dynamic way. It has been argued above that inaquaculture the microbial habitat undergoes continuous alter-ations, allowing constant changes in the structural compositionand the functions of the microbial community. It is unlikelythat a single bacterial species will be able to remain dominantin a continuously changing environment. The probability that abeneficial bacterium will dominate the associated microbiota ishigher when several bacteria are administered then when onlyone probiotic strain is involved.

At present, for a sample or situation, it is not feasible to

examine the extent to which all niches are filled in or, alterna-tively, how many opportunities are left for putative pathogensto grow and become a threat. Hence, one useful approach is tocarefully monitor shifts in the overall microbial communities.Methods which rapidly provide insights into how a communityof microbial species evolves are of utmost value. They maysignal instability and potential evolution of unwanted microbialassociations. Examples of such approaches are the monitoringof the Biolog pattern and the denaturing gradient gel electro-phores (DGGE) pattern (41, 60, 77, 78, 137). These ap-proaches, coupled with appropiate information technology,may permit the microbial ecology of aquaculture systems to bemonitored in the near future.

Techniques to indicate probiotic strains, such as the use ofgreen fluorescent protein or immunoassay procedure or 16S



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rDNA probes (70, 137), may also be useful in distinguishing anexogenous probiotc strain in a mixed microbial community, inorder to better locate and quantify the probiotic strain. Froma practical point of view, however, comparison of the spectrumof activity and the duration of the effect with those of antimi-crobials, vaccines, and immunostimulants should also be made.

In order to become of practical significance, probiotics ap-

plicable in large-scale aquaculture will have to be producedand formulated under industrial conditions that conform toquality control guidelines. Much effort obviously still has to beinvested in terms of the production of such multispecies inoc-ula and the methods needed to preserve them and to validatetheir quality.

REFERENCES

1. Atlas, R. M., and R. Bartha. 1997. Microbial ecologyfundamentals andapplications. Benjamin/Cummings Science Publishing, Menlo Park, Calif.

2. Austin, B., E. Baudet, and M. Stobie. 1992. Inhibition of bacterial fishpathogens by Tetraselmis suecica. J. Fish Dis. 15:5561.

3. Austin, B., and A. C. Billaud. 1990. Inhibition of the fish pathogen, Serratialiquefaciens, by an antibiotic-producing isolate of Planococcus recoveredfrom seawater. J. Fish Dis. 13:553556.

4. Austin, B., and J. G. Day. 1990. Inhibition of prawn p

probiotic bacteria as biological control agents

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