Probiotic for AquacultureImproving Health and PerformanceImproving Health and Performance

Inge KnapDirector, Research & Development, p

Chr. Hansen, Denmark

The research for probiotic for aquatic animals is increasingThe research for probiotic for aquatic animals is increasing with the demand for environment friendly aquaculture. Although application of probiotic in aquaculture seems to be relatively recent the interest in environment friendly treatments is increasing rapidly (Wang et al 2008)

Aquatic animals are quite different from land animals forAquatic animals are quite different from land animals for which most probiotic concepts have been developed.

Man and terrestrial livestock undergo embryonic development within an amnion, whereas the larval forms of most fish and h llfi h l d i th t l i t t lshellfish are released in the external environment at an early

ontogenetic stage. The larvae are highly exposed to gastrointestinal microbiota-associated disorders, because they g , ystart feeding even though the digestive tract is not fully developed and though the immune system is still incomplete (Gatesoupe 1999)(Gatesoupe 1999).

The digestive tract of hydrobionts is an open systemThe digestive tract of hydrobionts is an open system constantly in contact with the surrounding environment –water. Compared to water the digestive tract is an ecosystem far richer in nutrient and therefore more favourable for the growth of the majority of bacteria

This leads to both probiotic opportunities as well as pathogen challengeg

Introduction – Overview

P bi i f A lProbiotic for AquacultureImproving Health and Performance

Key criteria for use of probiotics in aquacultureWhat is a Probiotic?What kind of Probiotics are used?Improving the nutritional utilizationImproving the nutritional utilizationMode of action - Improving the nutritional utilizationDisease controlM d f ti Di t lMode of action – Disease controlBioremediationNew Probiotic

Key criteria for use of probiotics in aquacultureKey criteria for use of probiotics in aquaculture

Improving the nutritional utilizationImproving the nutritional utilizationIncreased weightFaster growthFaster growthLower feed conversion Better protein and fat efficiency rationp y

Disease control

Bioremediation

What is a Probiotic?What is a Probiotic?

The word “probiotic” was introduced by R P Parker in 1974 and his originalThe word probiotic was introduced by R.P. Parker in 1974 and his original definition was

“Organisms and substances which contribute to intestinal microbial balance”

R. Fuller revised the definition in 1989;

“Live microbial fed supplements which beneficially affects the host by improving its intestinalaffects the host by improving its intestinal microbial balance”

What kind of Probiotics are used?What kind of Probiotics are used?

Main used probioticsMain used probioticsLactic Acid Bacteria (LAB)Bacillus spBacillus spYeast

Other aquaculture related Probiotics but less common used;Pseudoalteromonas sp. Pseudomonas sp. and Vibrio fluvialis

Lactic acid bacteria (LAB)Lactic acid bacteria (LAB)

Definition of LABGram positive organismsMiroaerophilicFerment hexose sugars to primarily lactic acid

Lactococcus, Enterococcus, Pedicoccus, Streptococcus, Leuconostoc and Lactobacillus

LAB play a prominent role in the world food supplyPerform the main bio-conversations in fermented diary products, meats and vegetables .Critical for the production of silage, wine, coffee, sourdough, yoghurt and other fermented food products

LAB naturally associated to mucosal surfaces of animals and LAB’s belong to the normal microbiota of the gastrointestinal tract in healthy fish

All LAB are GRAS or has QPS status

Bacillus spBacillus sp.

Definition of BacillusGram positive organismsSpore formersBacillus is facultative aerobe but at present of nitrate/nitrite it can grow anaerobicBacillus is facultative aerobe but at present of nitrate/nitrite it can grow anaerobic

Bacillus as probiotic is in form of Bacillus sporesBacillus spores are

H bl bl H 2 3pH stable – stable at pH 2-3Trypsin stableSurvive stomach transitThermo-tolerant, survives pelletization <95°CStorage stable min 24 months when kept dry below 25°CStorage stable min. 24 months when kept dry below 25°CIn Feed stable >4 month stability in standard feed formulations

Bacillus spores germinate in the intestine, attach to mucus and multiply

Bacillus is found in soil and marine sediments

YeastYeast

Yeast are unicellular fungiYeast are unicellular fungiDominate fungal diversity in the oceans Large cells 20-300 times larger than bacteriaCell wall structure comprise β-glucansCell wall structure comprise β-glucans β-glucans – is used as immuno-stimulants

Yeast is used in the backing industry and for producing alcoholic beveragesYeast is used in the backing industry and for producing alcoholic beverages.

Yeast are used in ruminant feeding to reduce oxygen level in the rumen

Natural occurrence in healthy fish

D b h ii i “ lt l i ” t S di i t t iDebarryomyces hansenii is a “salt loving” yeast as Sodium is not toxic

Improving the nutritional utilizationImproving the nutritional utilization

Improving the nutritional utilizationImproving the nutritional utilizationIncreased weightFaster growthFaster growthLower feed conversion Better protein and fat efficiency rationp y

These effects of using probiotic is well documented within poultry, pigs, ruminant,

F t di i ltFew studies in aquaculture

Evaluation of Bacillus subtilish as a probiotic to Indian major Carp Labeo rohita (Kunar et al 2006)major Carp Labeo rohita (Kunar et al 2006)

Trial design:Trial design:12 cm long 15 ± 2g L. rohita100 liter tank with 70 l chlorine free tap water, 27°± 1°C, Hardness 82mg/l pH 7 4 dissolved oxygen 6 8 mg/l82mg/l, pH 7.4, dissolved oxygen 6.8 mg/l 2 weeks acclimatization followed with 15 days of treatment. 2 tanks pr. treatmentT T1 l T2 5E8 CFU T3 1E9 CFU T4 5E9 CFU fTreatment: T1= control, T2= 5E8 CFU, T3= 1E9 CFU, T4= 5E9 CFU of Bacillus subtilish

Results:Treatment T1

ControlT25E8 CFU

T31E9 CFU

T45E9 CFU

WG % 30.8a ±0.24 32.9b±0.37 34.8b±0.42 35.6c±0.8

Superscripts are significant different (P< 0.005p p g (

Improving the nutritional utilization

Other studies reported improved performance of usingOther studies reported improved performance of using probiotics:

Bacillus sp. improved length and weight of rainbow trout p p g g(Raida et al 2003)Growth and feed efficiency improved using the yeast S.

i i i I li (G t 2007)cerevisiae in Israeli carp (Gatesoupe 2007)The yeast Debaryomyces hansenii improved growth of larval from European sea bass (Gatesoupe 2007)from European sea bass (Gatesoupe 2007)Enterococcus faecium improve growth and feed efficiency of Israeli carp (Ringø 2004)

Mode of action - Improving the nutritional utilizationMode of action - Improving the nutritional utilization

MetabolitesBacillus is know to produce proteases, lipases and several carbohydrolases improving nutrient absorption (Ochoa-Solona and Olmos-Soto 2006)Gastrointestinal bacteria in hydrobionts take part in the decomposition of nutrients providing enzymes, amino acids and vitamins (Wang et al 2008)

Faster maturation of the intestineHigher brush border membrane enzymes activity after treatment with the yeast S. boulardii to rainbow trout fry – suggesting an earlier maturation of the digestive system (Waché et al 2006)

Reduce stressLowering cortisol level – Cortison is a hormon directly involved in the stress responses influencing metabolic and osmoregulatory pathways. Lb. delbrueckii showed such effect in of sea bass larvae (Silvi et al 2008)

Ensure healthy microbiota in the intestineSignificantly higher bacteria counts feeding Bacillus subtilish and Bacillus licheniformis to Nile tilapia fry (Shelby et al 2006)Lb. delbrueckii showed good capability to colonize the gut of sea bass larvae, therefore modifying the gut microbiota and exerting threefold higher effects on the survival of the treatedmodifying the gut microbiota and exerting threefold higher effects on the survival of the treated sea bass. (Silvi et al 2008)

Disease controlDisease control

Diseases caused by Vibrio spp, Yersinia spp. and Aeromonas spp. areDiseases caused by Vibrio spp, Yersinia spp. and Aeromonas spp. are commonly implicated in episodes of mortality in aquaculture

Faced with disease problems the common response has been to turnFaced with disease problems the common response has been to turn to antimicrobial drugs

Vaccination has been a success reducing the use of antibiotic in gsalmon production (95% reduction), but it is not always possible to use vaccine as a solution eg. shrimps have a non-specific immune response and vaccination can only provide short-term protection against pathogens (Hong et al 2005)against pathogens (Hong et al 2005)

Inhibition of pathogens via use of probiotics is an alternative solution.solution.

Most probiotic research in aquaculture deals with disease controls

Evaluation of Bacillus subtilis as a probiotic to Indian major Carp Labeo rohita (Kunar et al 2006)major Carp Labeo rohita (Kunar et al 2006)

Trial design:Trial design:12 cm long 15 ± 2g L. rohita100 liter tank with 70 l chlorine free tap water, 27°± 1°C, Hardness 82mg/l, pH 7.4, dissolved oxygen 6.8 mg/l 2 weeks acclimatization followed with 15 days of treatment + challenge with Aeromonas hydrophila. 2 tanks pr. treatmentTreatment: T1= control challenge, T2= 5E8 CFU, T3= 1E9 CFU, T4= 5E9 of Bacillus subtilis, CFU, T5= no challenge no treatment5E9 of Bacillus subtilis, CFU, T5 no challenge no treatment

Results:

Treatment T1 T2 T3 T4 T5Treatment T1 T2 T3 T4 T5

Survival % 18.75d ±6.25 68.75c ±6.25 81.25bc ±6.25 87.5ab ±0.00 100.00a

Superscripts are significant different (P< 0.005)

±0.00

p p g ( )

Disease control in shrimp aquaculture with probiotic bacteria (Moriarty 1999)probiotic bacteria (Moriarty 1999)

Vibrio harveyi have been implicated as the main bacterial pathogen ofVibrio harveyi have been implicated as the main bacterial pathogen of shrimpsVibrio sp. got resistant to every antibiotic used in the PhilippinesChlorine has been used in hatchery and ponds but it developed multiple y p p presistant bacteriaWhen probiotic Bacillus was used no disease was experienced and indeed survival was very high (80-100%)

Pond # 1Probiotic

2Probiotic

3Probiotic

4Control

5Control

6Control

Stocking 30 15 30 32 32 32gN/m2

Days of culture

195 189 196 182 190 120

Survival % 88 100 100 67 73 0

Harvest (tonne/ha)

7.2 5.4 8.6 6.5 6.8 0( )

Mode of action – disease controlMode of action – disease control

Immune stimulation Li et al (2007) improved immune response in white shrimp (Litopenaeus vannamei) – 3 types of haemocytes were significantly higher after treatment with Bacillus licheniformis

Toxin degradationBacillus Licheniformis has shown capability to reduce the effect of Clostridia perfringens toxin inBacillus Licheniformis has shown capability to reduce the effect of Clostridia perfringens toxin in chicken (Knap et al 2008)In a shrimp challenge model the mortality of Vibrio harveyi infection was significantly reduced by the presence of probiotic Bacillus in the tank water (Hong et al 2005)

Pathogen exclusionOne of the first theories for explaining effect of Probiotic and it is well documented in land animal species.

Some bacteria have antiviral effect The exact mechanism is not known Laboratory studiesSome bacteria have antiviral effect. The exact mechanism is not known. Laboratory studies indicate that extracellular agents from the bacteria inactivate the viruses (Wang et al 2008)

Bacterocin productionThe intestinal micro flora of hydrobionts play an important role in the resistance to infectious y p y pdiseases by producing antibacterial metabolites (Wang et al 2008)Bacillus is know to produce a variety of bacterocins:

Overview bacterocins in Bacillus

Urdaci & Pinchuk (2005)

Bioremediation

Aquaculture generates considerable amount of wastes:Aquaculture generates considerable amount of wastes:Metabolic by-productsResidual feedResidual feedFaecal matterResidues from prophylactic & therapeutic treatmentsp p y p

High concentration of these component generates stress and some could be toxic

Bi di ti i t i t lit b fBioremediation is a way to improve water quality by use of bacteria and enzymes

Use of bioremediationUse of bioremediation

Resulting in lower accumulation of slime or organic matters in the bottom of the pondbottom of the pondBetter penetration of oxygenBio-control – resulting in low pathogen pressure

Bacillus spp, Lactobacillus and Phenibacillus together with nitrifying bacteria have been reported to be key in this process

Bacillus spp is most often reported Fast multiplicationProducing high levels of extracellular enzyme complexesProducing high levels of extracellular enzyme complexesPathogen exclusionReduce total ammonium nitrogen

New ProbioticNew Probiotic

Many potential strains – easy to isolate from intestineMany potential strains easy to isolate from intestine

Focus should beCareful identificationPreferably GRAS or with QPS statusSafety – non patheticy pFree of transferable antibiotic resistanceScalable – and economic production processStorage stableStorage stableEasy to apply and use for end-user

Convincing efficacy documentationConsistent performance

Use of probiotic in the aquaculture industry is expanding forUse of probiotic in the aquaculture industry is expanding – for the benefit of the animal health and the economy in the aquaculture production.

Thank you for your attention