Lecture 7: Probiotic Bacteria

Lecture 7: Lecture 7: Probiotic Probiotic BacteriaBacteria

Lecture 7: Lecture 7: Probiotic Probiotic BacteriaBacteria

Lecture OutlineLecture Outline

Introduction/Definition of ProbioticsIntroduction/Definition of Probiotics Fundamental QuestionsFundamental Questions Recent FindingsRecent Findings Possible Modes of ActionPossible Modes of Action Rationale for Selecting ProbioticsRationale for Selecting Probiotics Conclusions and Further DirectionsConclusions and Further DirectionsSource of notes: Verschuere, L., Rombaut, G., Sorgeloos, P., and Verstraete, W., 2000. Probiotic bacteria as biological control agents in aquaculture. Microb. Mol. Biol. Rev., 64(4):655-671.

IntroductionIntroduction As we know, aquaculture is growing faster than As we know, aquaculture is growing faster than

beef cattle production (10% vs. 3%)beef cattle production (10% vs. 3%) Epizootics (disease) = major limiting factor in Epizootics (disease) = major limiting factor in

fish/shrimp developments!fish/shrimp developments! Disinfectants, antimicrobial drugs have had little Disinfectants, antimicrobial drugs have had little

effect in controlling disease.effect in controlling disease. Massive use of antimicrobials increases selective Massive use of antimicrobials increases selective

pressure on microbes and encourages natural pressure on microbes and encourages natural emergence of bacterial resistanceemergence of bacterial resistance

Resistant bacteria thrive after non-resistant Resistant bacteria thrive after non-resistant strains have been killed and can even pass on strains have been killed and can even pass on resistance genes to other bacteria that have not resistance genes to other bacteria that have not been exposed to antibioticsbeen exposed to antibiotics

IntroductionIntroduction Emphasis should be place on preventionEmphasis should be place on prevention More cost-effective than cure!More cost-effective than cure! Antimicrobials, disinfectants and pesticides Antimicrobials, disinfectants and pesticides

largely treat symptoms of the problem and largely treat symptoms of the problem and not not the causethe cause

Alternative strategies are just catching onAlternative strategies are just catching on ExampleExample:: via vaccination, Norway has reduced via vaccination, Norway has reduced

chemical therapeutant use from 50 MT in 1987 chemical therapeutant use from 50 MT in 1987 to less than 747 kg in 1997 (with 7x concomitant to less than 747 kg in 1997 (with 7x concomitant production increase!)production increase!)

OtherOther: use of immunostimulants w/or w/out : use of immunostimulants w/or w/out vaccinesvaccines

IntroductionIntroduction Use of bacteria as a food source and as a Use of bacteria as a food source and as a

biological control agent of fish disease was first biological control agent of fish disease was first proposed by Yasuda and Taga (1980)proposed by Yasuda and Taga (1980)

Vibrio alginolyticusVibrio alginolyticus has been used as a probiotic has been used as a probiotic in shrimp hatcheries in Ecuador since 1992in shrimp hatcheries in Ecuador since 1992

Reduced hatchery down time from 7 days per Reduced hatchery down time from 7 days per month to less than 21 days per year!month to less than 21 days per year!

FAO has now designated use of probiotics as a FAO has now designated use of probiotics as a major means for improvement of aquatic major means for improvement of aquatic environmental qualityenvironmental quality

Ultimate goalUltimate goal: make aquaculture products : make aquaculture products more acceptable to consumersmore acceptable to consumers

Definition of Definition of ProbioticsProbiotics

Many proposed, however: Many proposed, however: a live microbial feed a live microbial feed supplement which beneficially affects the host supplement which beneficially affects the host animal by improving its intestinal balanceanimal by improving its intestinal balance

Historically: terrestrial animals, genus Historically: terrestrial animals, genus LactobacillusLactobacillus

Definition (above) requires some additional Definition (above) requires some additional considerations:considerations:– 1) bacteria in aquatic medium influence composition of 1) bacteria in aquatic medium influence composition of

gut microbiota and vice versagut microbiota and vice versa– 2) immediate ambient environment has much greater 2) immediate ambient environment has much greater

influence on microbiota than with terrestrialsinfluence on microbiota than with terrestrials In aquatic environments, hosts and In aquatic environments, hosts and

microorganisms share the ecosystemmicroorganisms share the ecosystem Terrestrials: the gut represents a moist habitat Terrestrials: the gut represents a moist habitat

in a water-limited worldin a water-limited world

Definition of ProbioticsDefinition of Probiotics Big Issue 1Big Issue 1: : aquatics are surrounded by an environment aquatics are surrounded by an environment

supporting their pathogens independently of the host animalsupporting their pathogens independently of the host animal ResultResult: opportunistic pathogens can reach high densities : opportunistic pathogens can reach high densities

around the fish/shrimparound the fish/shrimp Surrounding bacteria are commonly ingested with the feed or Surrounding bacteria are commonly ingested with the feed or

via drinking (maximum case: filter feeders)via drinking (maximum case: filter feeders) Research in probiotics began with fish juveniles but more Research in probiotics began with fish juveniles but more

attention recently given to shrimp and finfish larvaeattention recently given to shrimp and finfish larvae Big Issue 2Big Issue 2: terrestrials have inherent colonizing bacteria : terrestrials have inherent colonizing bacteria

from the mother, aquatics largely spawned as axenic eggs (no from the mother, aquatics largely spawned as axenic eggs (no further contact with parent)further contact with parent)

Ambient bacteria colonize eggs surface, young larvae often Ambient bacteria colonize eggs surface, young larvae often have no developed gut (e.g., shrimp), no microbial community have no developed gut (e.g., shrimp), no microbial community in gut, gills or skinin gut, gills or skin

PointPoint: properties of bacteria in ambient water are very : properties of bacteria in ambient water are very importantimportant

Definition of ProbioticsDefinition of Probiotics Better definitionBetter definition: a live microbial adjunct which has a beneficial : a live microbial adjunct which has a beneficial

effect on the host by modifying the host-associated or ambient effect on the host by modifying the host-associated or ambient microbial community, by insuring improved use of feed or by microbial community, by insuring improved use of feed or by enhancing its nutrition, by enhancing the host response towards enhancing its nutrition, by enhancing the host response towards disease, or by improving quality of the ambient environmentdisease, or by improving quality of the ambient environment

Our focusOur focus: response towards disease and improvement of the : response towards disease and improvement of the ambient environmentambient environment

Jobs of Microbial Adjuncts:Jobs of Microbial Adjuncts:– 1) microbial adjuncts preventing proliferation of pathogens in gut or 1) microbial adjuncts preventing proliferation of pathogens in gut or

elsewhere;elsewhere;– 2) improved digestibility;2) improved digestibility;– 3) deliver improved nutrition to aquatics;3) deliver improved nutrition to aquatics;– 4) enhancing host response to disease (acquired);4) enhancing host response to disease (acquired);– 5) improved environmental quality.5) improved environmental quality.

Can you Manipulate Can you Manipulate Microbial Communities?Microbial Communities?

Difficult:Difficult: 1) discontinuous culture cycles; 2) 1) discontinuous culture cycles; 2) disinfection during pond prep; 3) sudden disinfection during pond prep; 3) sudden increases in nutrients due to feedingincreases in nutrients due to feeding

Unlikely under intensive rearingUnlikely under intensive rearing Must consider Must consider deterministic factorsdeterministic factors (known (known

response): salinity, temp, quality/quantity of response): salinity, temp, quality/quantity of feedfeed

Point: the environment selects the range of Point: the environment selects the range of microbes (axiom of environmental selection)microbes (axiom of environmental selection)

StochasticStochastic factorsfactors: chance, right place/right time: chance, right place/right time Evidence: identical cultures started Evidence: identical cultures started

simultaneously yield different assemblagessimultaneously yield different assemblages

Can you Manipulate Can you Manipulate Microbial Communities?Microbial Communities?

ConceptConcept: instead of accidental colonization, start with a : instead of accidental colonization, start with a probiotic that is well adapted to prevailing environmental probiotic that is well adapted to prevailing environmental conditionsconditions

This is probably better than competing with a dominant, well-This is probably better than competing with a dominant, well-established, non-probioticestablished, non-probiotic

Long-term exposure is often required to achieve a probiotic Long-term exposure is often required to achieve a probiotic effecteffect

Does the probiotic have to be continuously introduced to the Does the probiotic have to be continuously introduced to the culture?culture?

EvidenceEvidence: in most cases, yes (at least with : in most cases, yes (at least with Lactobacillus sp.Lactobacillus sp.)) Most fish contain a specific intestinal microbiota established at Most fish contain a specific intestinal microbiota established at

the juvenile stagethe juvenile stage Unless the host has been exposed to a limited range of Unless the host has been exposed to a limited range of

microorganisms in its development, a single addition won’t microorganisms in its development, a single addition won’t

result in long-term colonizationresult in long-term colonization

Recent FindingsRecent Findings As mentioned, it all started with Yasuda As mentioned, it all started with Yasuda

and Taga (1980);and Taga (1980); Usually added in feed or to culture water Usually added in feed or to culture water

as preventative agents against infection as preventative agents against infection by pathogenic bacteriaby pathogenic bacteria

Nutritional benefits are usually secondaryNutritional benefits are usually secondary Typical genera: Typical genera: LactobacillusLactobacillus,, VibrioVibrio,,

BacillusBacillus,, PseudomonasPseudomonas The following is a summary of findings The following is a summary of findings

based on various aquatic speciesbased on various aquatic species

Recent Findings: Recent Findings: fish fish eggs/larvaeeggs/larvae

For incubators, use of antibiotics must be minimalFor incubators, use of antibiotics must be minimal Antibiotics don’t represent control; instead, Antibiotics don’t represent control; instead,

unfavorable alteration of microbiotaunfavorable alteration of microbiota GoalGoal: establish colonization on the egg prior to : establish colonization on the egg prior to

pathogen colonization (i.e., no substrate)pathogen colonization (i.e., no substrate) This, in turn, affects subsequent gut colonizationThis, in turn, affects subsequent gut colonization Once initial feeding has started, probiotics typically Once initial feeding has started, probiotics typically

added to culture water or culture medium of live added to culture water or culture medium of live feed items (e.g., algae, rotifers, etc.)feed items (e.g., algae, rotifers, etc.)

ResultResult: improved survival, faster growth: improved survival, faster growth Mechanism? Production of antibiotics or Mechanism? Production of antibiotics or

siderophores (metal sequesterers)siderophores (metal sequesterers)

Recent Findings: Recent Findings: finfishfinfish

Digestive tract of finfish contains 10Digestive tract of finfish contains 1088 cells/g (Ringo et cells/g (Ringo et al., 1995)al., 1995)

For cod, Gadus gadus, gut is colonized by similar For cod, Gadus gadus, gut is colonized by similar bacteria as found in eggs (Hansen and Olafsen, 1999)bacteria as found in eggs (Hansen and Olafsen, 1999)

Putative probiotics added as soon as possible after Putative probiotics added as soon as possible after hatching in order to colonize gut prior to feeding hatching in order to colonize gut prior to feeding (Ringo and Vadstein, 1998)(Ringo and Vadstein, 1998)

Turbot and dab harbor bacteria capable of suppressing Turbot and dab harbor bacteria capable of suppressing growth of growth of VV. anguillarum (Ollson et al., 1992). anguillarum (Ollson et al., 1992)

VV. . alginolyticusalginolyticus was effective in reducing disease was effective in reducing disease caused by caused by AeromonasAeromonas salmonicidasalmonicida in Atlantic salmon in Atlantic salmon (Austin et al., 1995)(Austin et al., 1995)

Recent Findings: Recent Findings: finfish finfish

Kennedy et al. (1998) showed addition of a Gram-positive Kennedy et al. (1998) showed addition of a Gram-positive probiotic increased survival, size uniformity, and growth rate probiotic increased survival, size uniformity, and growth rate of snook, red drum, spotted sea trout and striped mullet.of snook, red drum, spotted sea trout and striped mullet.

Gram et al. (1999) reported a strain of Gram et al. (1999) reported a strain of PseudomonasPseudomonas fluorescensfluorescens reduced mortality of 40 g rainbow trout infected reduced mortality of 40 g rainbow trout infected with pathogenic with pathogenic VV. . anguillarumanguillarum

Garcia-de-la-Banda et al. (1992) added Garcia-de-la-Banda et al. (1992) added StreptococcusStreptococcus lactis lactis and Lactobacillus bulgaricus to rotifers and and Lactobacillus bulgaricus to rotifers and ArtemiaArtemia sp. sp. nauplii and recorded 6x higher survival at weaning than nauplii and recorded 6x higher survival at weaning than untreated controlsuntreated controls

Nikoskelainen et al. (2003) showed immune enhancement in Nikoskelainen et al. (2003) showed immune enhancement in rainbow trout via rainbow trout via Lactobacillus rhamnosusLactobacillus rhamnosus supplemented in supplemented in feedsfeeds

Recent Findings: Recent Findings: shrimpshrimp Broad application in hatcheries, but few in-depth studies publishedBroad application in hatcheries, but few in-depth studies published often used as food source (e.g., soil bacteria for often used as food source (e.g., soil bacteria for P. monodon P. monodon

nauplii)nauplii) improved survival (57% vs. 0%) after 13 days against improved survival (57% vs. 0%) after 13 days against V. V.

anguillarumanguillarum improved survival of improved survival of L. vannamei L. vannamei PL’s inoculated with PL’s inoculated with V. V.

alginolyticusalginolyticus (non-pathogenic) vs. oxytet and control (Garriques (non-pathogenic) vs. oxytet and control (Garriques and Arivalo, 1995)and Arivalo, 1995)

Griffith (1995) reported that following the introduction of probiotics Griffith (1995) reported that following the introduction of probiotics in Ecuador in 1992, hatchery down-time between batches was in Ecuador in 1992, hatchery down-time between batches was reduced from 7 days per month to 21 days per year, production reduced from 7 days per month to 21 days per year, production volumes increased by 35% and antimicrobial use decreased by volumes increased by 35% and antimicrobial use decreased by 94%94%

In shrimp hatcheries in New Caledonia, a strain of In shrimp hatcheries in New Caledonia, a strain of Pseudoalteromonas piscicida Pseudoalteromonas piscicida was found to inhibit the growth of was found to inhibit the growth of VibrioVibrio sp. (Saulnier et al., 2000) sp. (Saulnier et al., 2000)

Recent Findings: Recent Findings: bivalave molluscsbivalave molluscs

Most research has focused on nutritional Most research has focused on nutritional contributions to mollusc larvaecontributions to mollusc larvae

most work most work in vitroin vitro wherein autochthonous wherein autochthonous strains have been isolated from scallops strains have been isolated from scallops and have shown some inhibition to and have shown some inhibition to Vibrio Vibrio sp.sp. a and nd Aeromonas hydrophilaAeromonas hydrophila

Bacillus sp. Bacillus sp. and and Lactobacillus sp.Lactobacillus sp. shown to shown to depurate oysters (depurate oysters (Crassostrea virginicaCrassostrea virginica) ) against against V. vulnificus V. vulnificus (Williams et al., 2001) (Williams et al., 2001)

Part II. Modes of Part II. Modes of Action of Action of Probiotic Probiotic

BacteriaBacteria

Modes of ActionModes of Action Many publications about probiotics have Many publications about probiotics have

emerged in aquaculture in the last decadeemerged in aquaculture in the last decade most based on empirical arguments most based on empirical arguments

(simple)(simple) modes of action were largely modes of action were largely

circumstantialcircumstantial several modes of action have been several modes of action have been

proposed as a result of human and proposed as a result of human and agricultural applicationsagricultural applications

Modes of ActionModes of Action Info on terrestrial investigations has been used for Info on terrestrial investigations has been used for

aquatics (esp. aquatics (esp. Lactobacillus sp.Lactobacillus sp.))

one problem: distinction between 1) the intrinsic one problem: distinction between 1) the intrinsic ability of the ability of the probingprobing to positively influence the to positively influence the host and 2) its ability to reach and maintain itself in host and 2) its ability to reach and maintain itself in the location where the effect is to be exertedthe location where the effect is to be exerted

PointPoint: does not matter if it produces : does not matter if it produces siderophoressiderophores or inhibitory compounds in the gut if it’s never or inhibitory compounds in the gut if it’s never ingestedingested

can be very important: can be very important: Mytilus edulis Mytilus edulis (Blue (Blue Muscle)Muscle) can selectively ingest/digest microbescan selectively ingest/digest microbes

Modes of ActionModes of Action

Also, if the candidate probiont Also, if the candidate probiont cannot proliferate in the gut it cannot proliferate in the gut it probably won’t exert a strong effectprobably won’t exert a strong effect

will need will need continuous applicationcontinuous application through the diet or via the water ($through the diet or via the water ($$$)$$)

summarysummary: they must reach the : they must reach the location where effect is desiredlocation where effect is desired

Possible Modes of Possible Modes of ActionAction

production of inhibitory compoundsproduction of inhibitory compounds competition for chemicals/available competition for chemicals/available

energyenergy competition for adhesion sites (exclusion)competition for adhesion sites (exclusion) enhancement of the immune responseenhancement of the immune response improvement of water qualityimprovement of water quality interaction with phytoplanktoninteraction with phytoplankton a source of macro- and micro-nutrientsa source of macro- and micro-nutrients enzymatic contribution to digestion enzymatic contribution to digestion

(1) production of (1) production of inhibitory compoundsinhibitory compounds Release of chemicals having a bactericidal or Release of chemicals having a bactericidal or

bacteriostatic effectbacteriostatic effect ultimate result: competitive edge for ultimate result: competitive edge for

nutrients/energynutrients/energy production sites:production sites: in host intestine, on its in host intestine, on its

surface, or in culture mediumsurface, or in culture medium products:products: antibiotics, bacteriocins, antibiotics, bacteriocins,

siderophores, lysozymes, proteases, siderophores, lysozymes, proteases, hydrogen peroxide, organic acids (pH change)hydrogen peroxide, organic acids (pH change)

exact compound is seldom identified: hence, exact compound is seldom identified: hence, the term the term “inhibitory”“inhibitory”

(1) production of (1) production of inhibitory compoundsinhibitory compounds Lactobacillus sp.Lactobacillus sp. produces bacteriocins (toxins)produces bacteriocins (toxins) marine bacteria produce bacteriolytic enzymes marine bacteria produce bacteriolytic enzymes

against against V. parahaemolyticusV. parahaemolyticus Alteromonas sp.Alteromonas sp. produces produces monastatinmonastatin, shown , shown

to be inhibitory against to be inhibitory against Aeromonas hydrophilaAeromonas hydrophila inhibitory effects have been shown by inhibitory effects have been shown by

probiotics against aquaculture pathogensprobiotics against aquaculture pathogens no demonstration under no demonstration under in vivoin vivo conditions conditions

(oops!)(oops!) more research required!!!more research required!!! (Didn’t you mention this last time??)(Didn’t you mention this last time??)

(2) Competition for (2) Competition for Chemicals or Available Chemicals or Available

EnergyEnergy Explains how different microbial populations exist in Explains how different microbial populations exist in

same ecosystemsame ecosystem it is likely that it occurs in the mammalian gut, but it is likely that it occurs in the mammalian gut, but

proof is lackingproof is lacking application of the principles of competition to natural application of the principles of competition to natural

situations is not easysituations is not easy microbial situation in ecosystems is usually microbial situation in ecosystems is usually controlled controlled

by heterotrophsby heterotrophs competing for organic substrates as competing for organic substrates as both carbon and energy sourcesboth carbon and energy sources

if you know the factors affecting microbial composition if you know the factors affecting microbial composition of the microbiota, of the microbiota, you can manipulate ityou can manipulate it

(2) Competition for (2) Competition for Chemicals or Available Chemicals or Available

EnergyEnergy All microorganisms require iron for growthAll microorganisms require iron for growth siderophore:siderophore: low mw ferric ion-specific chelating low mw ferric ion-specific chelating

agentsagents dissolve precipitated Fe and make it available for dissolve precipitated Fe and make it available for

microbial growthmicrobial growth siderophores scavenge Fe and make it unavailable siderophores scavenge Fe and make it unavailable

to other speciesto other species this occurs at tissue levelthis occurs at tissue level probiotics producing siderophores can outcompete probiotics producing siderophores can outcompete

pathogens for Fe, thus limiting pathogen growthpathogens for Fe, thus limiting pathogen growth works best with pathogens that also produce works best with pathogens that also produce

siderophores (e.g., siderophores (e.g., V. anguillarumV. anguillarum))

(3) Competition for (3) Competition for Adhesion SitesAdhesion Sites

Competition for gut adhesion sites would limit Competition for gut adhesion sites would limit colonizationcolonization

adhesion to enteric mucus is necessary for adhesion to enteric mucus is necessary for bacteria to become established in fish intestinesbacteria to become established in fish intestines

this is probably the first probiotic effectthis is probably the first probiotic effect adhesion can be specific (based on adhesin and adhesion can be specific (based on adhesin and

receptor molecules) or non-specific (based on receptor molecules) or non-specific (based on physiochemical factors)physiochemical factors)

total probiotic effect is probably a mixture of total probiotic effect is probably a mixture of site competition, production of inhibitory site competition, production of inhibitory compounds and nutrient/energy competitioncompounds and nutrient/energy competition

(4) Enhancement of (4) Enhancement of Immune ResponseImmune Response

Rem definition of an Rem definition of an immunostimulantimmunostimulant? ? Chemical Chemical compounds that activate the immune systems of compounds that activate the immune systems of animals and render them more resistant to animals and render them more resistant to infections by viruses, bacteria, fungi and infections by viruses, bacteria, fungi and parasites.parasites.

Immune response varies in animalsImmune response varies in animals lactic acid bacteria administered orally may lactic acid bacteria administered orally may

induce increased resistance to enteric infections induce increased resistance to enteric infections problem: only shown with specific cell problem: only shown with specific cell

compounds or dead cellscompounds or dead cells good indications, but no proofgood indications, but no proof

(5) Improvement of (5) Improvement of Water QualityWater Quality

Proposed as a mode of action as a result of Proposed as a mode of action as a result of monitoring water quality after addition of monitoring water quality after addition of probioticsprobiotics

usually associated with usually associated with Bacillus sp.Bacillus sp. HookHook: gram + bacteria are better converters : gram + bacteria are better converters

of organic matter back to COof organic matter back to CO22 than gram - than gram - thusthus: phytoplankton blooms are more easily : phytoplankton blooms are more easily

maintained (interesting research area!)maintained (interesting research area!) monitormonitor: DOC, POC: DOC, POC so far, hasn’t been demonstratedso far, hasn’t been demonstrated

Rationale for Rationale for Selecting Selecting ProbioticsProbiotics

Verschuere et al. (2000)

Background InfoBackground Info

Profound knowledge of culture Profound knowledge of culture Critical review of available literatureCritical review of available literature Characterization of abiotic and biotic Characterization of abiotic and biotic

environment impacting cultureenvironment impacting culture Must know relationship between Must know relationship between

microbiota and host well-understoodmicrobiota and host well-understood Relationship between microbiota and Relationship between microbiota and

carrying capacity of environmentcarrying capacity of environment

Acquisition of Putative Acquisition of Putative ProbioticsProbiotics

Are Are putativeputative probiotics (isolated from host or probiotics (isolated from host or host environment) better than those from other host environment) better than those from other sources?sources?

Putatives might be better due to fact they are Putatives might be better due to fact they are already adhered to gut wall (i.e., could block already adhered to gut wall (i.e., could block pathogen adhesion)pathogen adhesion)

If a non-pathogenic bacterium exists at high If a non-pathogenic bacterium exists at high density in typical culture water, it will be well-density in typical culture water, it will be well-adapted to prevailing conditions and can adapted to prevailing conditions and can probably compete efficiently with pathogens probably compete efficiently with pathogens for nutrientsfor nutrients

Screening of Putatives:Screening of Putatives: In-vitro In-vitro antagonism antagonism

testingtesting

Candidates are exposed to pathogen in Candidates are exposed to pathogen in liquid or solid mediumliquid or solid medium

screened for production of inhibitory screened for production of inhibitory compounds (bacteriocins), siderophores, or compounds (bacteriocins), siderophores, or on competition for nutrientson competition for nutrients

must be undertaken with extreme caution: must be undertaken with extreme caution: not all media are suitablenot all media are suitable

probionts are finicky as to on which medium probionts are finicky as to on which medium they produce inhibitory compounds (e.g., they produce inhibitory compounds (e.g., marine agar vs. TSB)marine agar vs. TSB)

Evaluation of Evaluation of PathogenicityPathogenicity

Probiotics must not be pathogenic to the host organism Probiotics must not be pathogenic to the host organism -- this must be confirmed prior to acceptance-- this must be confirmed prior to acceptance

host must be challenged under stressed and non-host must be challenged under stressed and non-stressed conditionsstressed conditions

usually accomplished by adding probiotic to the culture usually accomplished by adding probiotic to the culture waterwater

proper way to do this under monoxenic conditions (only proper way to do this under monoxenic conditions (only the probiont present)the probiont present)

also look at interaction with other food organisms found also look at interaction with other food organisms found simultaneously in culture (e.g., algae)simultaneously in culture (e.g., algae)

In-vivoIn-vivo Evaluations Evaluations Involves introducing candidate species to Involves introducing candidate species to

host cultures and monitoring growth, host cultures and monitoring growth, survival, physiochemical parameterssurvival, physiochemical parameters

means of additionmeans of addition: addition to artificial diet, : addition to artificial diet, addition to culture water, bathing, addition addition to culture water, bathing, addition via live foodvia live food

next stepnext step: experimental (allochthonous) : experimental (allochthonous) infection of host via immersioninfection of host via immersion

needs long-term evaluation (is the needs long-term evaluation (is the pathogenic effect one of suppression or pathogenic effect one of suppression or delay?)delay?)

Conclusions/FutureConclusions/Future Current status of probiotics in Current status of probiotics in

aquaculture is really hazyaquaculture is really hazy Lack of knowledge on modes of actions Lack of knowledge on modes of actions

is very evidentis very evident more info on competitive processes more info on competitive processes

between bacteria is requiredbetween bacteria is required more info on relationship between more info on relationship between

bacteria and other microbiota requiredbacteria and other microbiota required economic value/efficiency, anyone???economic value/efficiency, anyone???