Probiotic and Prebiotic Probiotic and Prebiotic Application in ChickensApplication in Chickens

J. Stan BaileyJ. Stan Bailey

USDA, ARS, BEARUSDA, ARS, BEARAthens, GAAthens, GA

stan.bailey@ars.usda.govstan.bailey@ars.usda.gov

Why the interest in proWhy the interest in pro-- and and prebiotics use in chickens?prebiotics use in chickens?

Pathogen control (particularly Pathogen control (particularly SalmonellaSalmonella))Improved health and associated Improved health and associated production performance enhancementproduction performance enhancementPressure to reduce antibiotic use in Pressure to reduce antibiotic use in animal agricultureanimal agriculture

Broiler Production 101Broiler Production 101

Days 0-21

Days 42-56

Consumption

15,000 to 40,000 per house

Growout

HatcheryTransport

&ProcessingDays 42-56

http://www.jefo.ca/pdf/Intestinal_Health.pdf

Gastrointestinal TractGastrointestinal TractLargest immune organ in the bodyLargest immune organ in the body•• (Abreu(Abreu--Targan, 1996; Mayer, 2000)Targan, 1996; Mayer, 2000)

Underdeveloped in poultry at hatchUnderdeveloped in poultry at hatch•• (Uni et al.,1995; Noy and Sklan, 1997)(Uni et al.,1995; Noy and Sklan, 1997)

Microflora aid in many aspects of Microflora aid in many aspects of maturation: assembly of GALT, increases maturation: assembly of GALT, increases integrity of mucosal barrier, integrity of mucosal barrier, ‘‘educateseducates’’immune systemimmune system•• (Cebra et al., 1999; MacPherson et al., 2000; (Cebra et al., 1999; MacPherson et al., 2000;

Kelly et al., 2004)Kelly et al., 2004)

Intestinal Ecosystem is Complex and Dynamic

From Jiangrang et al., 2003

Chicken microflora determined by sequencing of 1,230 clones from a 16S rDNA community DNA library.

Human salmonellosisHuman salmonellosisEstimated that 1.4 million people contract Estimated that 1.4 million people contract salmonellosis in the U.S. annuallysalmonellosis in the U.S. annually•• $3,000,000,000 estimated annual costs (WHO $3,000,000,000 estimated annual costs (WHO

2006)2006)In 2004 In 2004 SalmonellaSalmonella was associated with 42% of all was associated with 42% of all laboratory confirmed food borne illnesseslaboratory confirmed food borne illnesses•• (FoodNet 2005)(FoodNet 2005)

Poultry products serve as vehicle for human Poultry products serve as vehicle for human salmonellosis salmonellosis •• (Bean & Griffin, 1990; Persson & Jendteg 1992)(Bean & Griffin, 1990; Persson & Jendteg 1992)

Pathogen controlPathogen control

Chickens are most Chickens are most susceptible to susceptible to colonization by colonization by SalmonellaSalmonella when when they are newly they are newly hatched or after hatched or after times of stress times of stress (immature and/or (immature and/or disrupted intestinal disrupted intestinal microflora)microflora)

CDCD--5050•• Day 1 Day 1 –– 101000

•• Day 3 Day 3 –– 101022--33

•• Day 7 Day 7 –– 101066--77

StressStress•• TemperatureTemperature•• Subclinical diseaseSubclinical disease•• TransportTransport•• MoltingMolting

Improved health and performanceImproved health and performance

Improved weight gain and feed Improved weight gain and feed conversionconversionHelp prevent and overcome stress Help prevent and overcome stress during productionduring productionImprove birds ability to Improve birds ability to immunologically respond to challengesimmunologically respond to challenges

Reduced antibiotic useReduced antibiotic useAntibiotics are used either therapeutically to Antibiotics are used either therapeutically to treat a disease condition or treat a disease condition or subtherapeutically to enable the chickens to subtherapeutically to enable the chickens to reach their full growth potential by reducing reach their full growth potential by reducing subclinical disease and/or selection of more subclinical disease and/or selection of more productive gut microfloraproductive gut microfloraReduced subtherapeutic use is leading to Reduced subtherapeutic use is leading to increased necrotic enteritis (often caused by increased necrotic enteritis (often caused by Clostridium perfringens)Clostridium perfringens)PrePre-- and probiotics may be an alternative and probiotics may be an alternative

DENMARK DENMARK –– Increases in Therapeutic Increases in Therapeutic Use of Antimicrobials Since the Bans on Use of Antimicrobials Since the Bans on

AGPsAGPs

4800057000 57300 61900

8060091602 96960 102500

112500 112650

40000

60000

80000

100000

120000

1996 1997 1998 1999 2000 2001 2002 2003 2004 2005

Source: DANMAP 1996-2005

Subtherapeutic ban

ProbioticProbiotic..

A direct fed microbial which alters A direct fed microbial which alters the microflora of the host and exert the microflora of the host and exert beneficial effects to the hostbeneficial effects to the host•• Viable includes both defined and Viable includes both defined and

undefinedundefined•• Beneficial includes both health benefit to Beneficial includes both health benefit to

the animal and indirect benefit to the animal and indirect benefit to human health such as reduction of human health such as reduction of pathogenic bacteria in/on the animalpathogenic bacteria in/on the animal

Characteristics of ideal ProbioticsCharacteristics of ideal ProbioticsPatterson and Burkholder, 2003 Patterson and Burkholder, 2003

adapted from Simmering and Blaut, 2001adapted from Simmering and Blaut, 2001

Be of host originBe of host originNonNon--pathogenicpathogenicWithstand processing and storageWithstand processing and storageResist gastric acid and bileResist gastric acid and bileAdhere to epithelium or mucusAdhere to epithelium or mucusPersist in the intestinal tractPersist in the intestinal tractProduce inhibitory compoundsProduce inhibitory compoundsModulate immune responseModulate immune responseAlter microbial activitiesAlter microbial activities

Mode of Action of ProbioticsMode of Action of ProbioticsJin et al., 1997, World Poultry ScienceJin et al., 1997, World Poultry Science

Maintain normal intestinal microflora by Maintain normal intestinal microflora by competitive exclusion and antagonismcompetitive exclusion and antagonismAlter metabolism by increasing digestive Alter metabolism by increasing digestive enzyme activity and decreasing bacterial enzyme activity and decreasing bacterial enzyme activity and ammonia productionenzyme activity and ammonia productionImprove feed intake and digestionImprove feed intake and digestionNeutralize enterotoxins and stimulate the Neutralize enterotoxins and stimulate the immune systemimmune system

PrebioticPrebiotic

A nondigestible food ingredient that A nondigestible food ingredient that affects the host by selectively affects the host by selectively stimulating the growth and/or stimulating the growth and/or activity of beneficial bacteria in the activity of beneficial bacteria in the intestinal tract.intestinal tract.•• Beneficial bacteria could provide both a Beneficial bacteria could provide both a

health benefit to the animal and indirect health benefit to the animal and indirect benefit to human health such as benefit to human health such as reduction of pathogenic bacteria in/on reduction of pathogenic bacteria in/on the animalthe animal

Characteristics of ideal PrebioticsCharacteristics of ideal PrebioticsPatterson and Burkholder, 2003 Patterson and Burkholder, 2003

adapted from Simmering and Blaut, 2001adapted from Simmering and Blaut, 2001

Be neither hydrolyzed nor absorbed Be neither hydrolyzed nor absorbed by mammalian enzymes or tissuesby mammalian enzymes or tissuesSelectively enrich for one or a limited Selectively enrich for one or a limited number of beneficial bacterianumber of beneficial bacteriaBeneficially alter the intestinal Beneficially alter the intestinal microbiota and their activitiesmicrobiota and their activitiesBeneficially alter luminal or systemic Beneficially alter luminal or systemic aspects of the host defense systemaspects of the host defense system

ProbioticsProbiotics

Probiotics Probiotics -- DefinedDefined–Known species of flora

–Lactic acid bacteria (LAB): Lactobacillus spp,

Lactobacillus reuteri, Weisiella spp, Bifidobacterium spp,

Enterococcus spp, Lactococcus spp, Pediococcus spp;

Streptococcus thermophilus, (or various combinatios)

–non-LAB group:

>Bacillus cereus and B. licheniformis

>Saccharomyces spp.

Probiotics Probiotics -- UndefinedUndefined

Normal intestinal microflora from Normal intestinal microflora from healthy adult chicken (animal)healthy adult chicken (animal)Wider diversity of microorganismsWider diversity of microorganismsAppears to be more effective than Appears to be more effective than defined organismsdefined organismsBroilactBroilact™™, Aviguard, Aviguard™™, Mucosal , Mucosal Stater CultureStater Culture™™

Early Probiotic WorkEarly Probiotic Work

Early colonization with normal Early colonization with normal

microflora improved resistance to microflora improved resistance to

bacterial pathogens bacterial pathogens (Nurmi (Nurmi et al.et al., 1972), 1972)

Increased productivity of poultry (>75 Increased productivity of poultry (>75

studies)studies)

Effects are doseEffects are dose--related, isolate related, isolate

specific, and require viable bacteriaspecific, and require viable bacteria

Nurmi, E.V. and Rantala M. 1973. Prevention of the growth of Nurmi, E.V. and Rantala M. 1973. Prevention of the growth of SalmonellaSalmonella infantisinfantis in chicks by the flora of the alimentary tract in chicks by the flora of the alimentary tract of chickens. Brit. Poult. Sci. 14(6): 627of chickens. Brit. Poult. Sci. 14(6): 627--630.630.Barrow, P.A. and J.F. Tucker. 1986. Inhibition of colonization Barrow, P.A. and J.F. Tucker. 1986. Inhibition of colonization of the chicken caecum with of the chicken caecum with Salmonella typhimuriumSalmonella typhimurium by preby pre--treatment with strains of treatment with strains of Escherichia coliEscherichia coli. Journal of Hygiene, . Journal of Hygiene, Camb. 96:161Camb. 96:161--169.169.Blankenship, L.C., J.S. Bailey, N.A. Cox, N.J. Stern, R. Brewer,Blankenship, L.C., J.S. Bailey, N.A. Cox, N.J. Stern, R. Brewer,and O. Williams. 1993. Twoand O. Williams. 1993. Two--step mucosal competitive exclusion step mucosal competitive exclusion flora treatment to diminish flora treatment to diminish salmonellaesalmonellae in commercial broiler in commercial broiler chickens. Poultry Science. 72:1667chickens. Poultry Science. 72:1667--1672.1672.Corrier, D.E., D.J. Nisbet, C.M. Scanlan, A.G. Hollister, and Corrier, D.E., D.J. Nisbet, C.M. Scanlan, A.G. Hollister, and J.R. DeLoach. 1995. Control of J.R. DeLoach. 1995. Control of Salmonella typhimuriumSalmonella typhimuriumcolonization in broiler chicks with a continuouscolonization in broiler chicks with a continuous--flow flow characterized mixed culture of cecal bacteria. Poultry Science. characterized mixed culture of cecal bacteria. Poultry Science. 74:91674:916--924924

19721972--1995 Emphasis on Prophylaxis1995 Emphasis on Prophylaxis

Recent (10 year) Probiotic WorkRecent (10 year) Probiotic WorkAn large number of scientific publications An large number of scientific publications on Lactic Acid Bacteria and human health on Lactic Acid Bacteria and human health model studiesmodel studiesReports of benefits for enteric bacterial & Reports of benefits for enteric bacterial & viral diseases, reduced effects of viral diseases, reduced effects of mycotoxins, cancer intervention, increased mycotoxins, cancer intervention, increased absorption of macrominerals and other absorption of macrominerals and other nutrients. nutrients. Reid and Friendship. Animal Biotech: Reid and Friendship. Animal Biotech: 13:9713:97--112, 2002. Excellent review of 112, 2002. Excellent review of probiotics use in animals including poultry.probiotics use in animals including poultry.

Example ReferencesExample References-- Immune ResponsesImmune Responses

Fukushima Y. Fukushima Y. et al.et al. (1998) (1998) Int. J. Food Int. J. Food Microbiol.Microbiol.42:3942:39--44.44.

TejadaTejada--Simon MV Simon MV et al.et al. (1999) (1999) J. Dairy Sci. J. Dairy Sci. 82:64982:649--660.660.

Fang H Fang H et al.et al. (2000) (2000) FEMS Immunol. Med. FEMS Immunol. Med. MicrobiolMicrobiol. 29:47. 29:47--52.52.

Cukrowska B. Cukrowska B. et al.et al. (2002) (2002) Scand. J. Scand. J. Immunol. Immunol. 55:20455:204--209209..

Reported Effects of Probiotics on Reported Effects of Probiotics on Laying HensLaying Hens

Increasing egg Increasing egg productionproduction(Nahashon (Nahashon et al.et al. 1996)1996)

Increasing egg shell Increasing egg shell qualityquality(Mohan, B. (Mohan, B. et al. et al. 1995) 1995)

Improved Feed Improved Feed ConversionConversion(Yeo and Kim, 1997)(Yeo and Kim, 1997)

Decreasing MortalityDecreasing Mortality(Kumprecht and Zobac, (Kumprecht and Zobac, 1998)1998)

Decreasing Cost of Decreasing Cost of ProductionProduction((Jin, L. Jin, L. et al. et al. 1999)1999)

Growth Performance and Growth Performance and ProbioticsProbiotics

L. acidophilusL. acidophilus added to drinking water added to drinking water increased weight gain by 10% (Tortuero, increased weight gain by 10% (Tortuero, 1973)1973)B. coagulansB. coagulans in the feed d 1in the feed d 1--49 increased 49 increased mean body weight (Cavazzoni et. al., 1998)mean body weight (Cavazzoni et. al., 1998)2 2 LactobacilliLactobacilli in feed increased body weights in feed increased body weights by approximately 10% (d30, 35, and 40) by approximately 10% (d30, 35, and 40) (Lan et al., 2003)(Lan et al., 2003)

Immune Stimulation by Immune Stimulation by ProbioticsProbiotics

Strains differ in ability to change cytokine profiles Strains differ in ability to change cytokine profiles (Perdigon et al., 2002; Maasen et al., 2000; Dalloul et (Perdigon et al., 2002; Maasen et al., 2000; Dalloul et al., 2005; Vinderola et al., 2004)al., 2005; Vinderola et al., 2004)Strains differ in ability to increase immunoglobulins Strains differ in ability to increase immunoglobulins (Yurong et al., 2005; Dalloul et al., 2005; Vinderola (Yurong et al., 2005; Dalloul et al., 2005; Vinderola et al., 2004)et al., 2004)Coincubation of 2 individual LAB with or without ST Coincubation of 2 individual LAB with or without ST inhibit ILinhibit IL--8, but don8, but don’’t inhibit binding of ST to cell t inhibit binding of ST to cell culture (Oculture (O’’Hara et al., 2006)Hara et al., 2006)

PrebioticsPrebiotics

Prebiotics Prebiotics -- CharacteristicsCharacteristics

NonNon--digestible by the hostdigestible by the host

Selective growth stimulationSelective growth stimulation

Modulates bacterial population in the Modulates bacterial population in the

GITGIT

Brings beneficial health effects to the Brings beneficial health effects to the

hosthost

Particularly for poultry production: Particularly for poultry production:

low dietary inclusion rateslow dietary inclusion rates !!

Prebiotics Prebiotics -- TypesTypesCarbohydratesCarbohydrates•• Resistant starchResistant starch•• Dietary fibre (nonDietary fibre (non--starch polysaccharides)starch polysaccharides)

pectin, cellulose, hemicellulose, xilan, and guar gumpectin, cellulose, hemicellulose, xilan, and guar gum

•• OligosaccharidesOligosaccharideslactose, lactulose, raffinose, stachyose, fructolactose, lactulose, raffinose, stachyose, fructo--, gluco, gluco--, and , and mannanmannan--oligosaccharides*oligosaccharides*

Some protein and aminoacidsSome protein and aminoacids•• endogenous and bacterial secretionsendogenous and bacterial secretions

Why lactose as prebiotic?Why lactose as prebiotic?

Fermented preferentially by LAB Fermented preferentially by LAB →→

selectiveselective

Non digested by poultry speciesNon digested by poultry species

•• No presence of endogenous lactase No presence of endogenous lactase

considered considered ((Denbow, 2000Denbow, 2000 ))

Versatile product: feed or drinking waterVersatile product: feed or drinking water

Lactose Studies in PoultryLactose Studies in Poultry

Focused mainly on control of Focused mainly on control of pathogenspathogens

Few studies focused on performanceFew studies focused on performance

• The main problem:

High doses: 2 % and up

Prebiotic inclusion rates in feed: < 1 %

USDA, ARS Stan Bailey USDA, ARS Stan Bailey prebiotic, probiotic researchprebiotic, probiotic research

FOS (fructooligosaccharide)FOS (fructooligosaccharide)Yeast (Saccharomyces boulardii)Yeast (Saccharomyces boulardii)Competitive ExclusionCompetitive Exclusion

Effect of FOS on Effect of FOS on SalmonellaSalmonella colonization colonization of chickens (of chickens (Bailey et al., 1991)Bailey et al., 1991)

As prebiotic gave limited (12%) improveAs prebiotic gave limited (12%) improve--ment in ment in Salmonella Salmonella colonization ratescolonization ratesImproved efficacy of CE culture (3 fold)Improved efficacy of CE culture (3 fold)When chickens were stressed the FOS When chickens were stressed the FOS reduced Salmonella colonization from 92% reduced Salmonella colonization from 92% to 25%to 25%

Saccharomyces boulardiiSaccharomyces boulardii(Line, Bailey et al., 1998)(Line, Bailey et al., 1998)

0

10

20

30

40

50

60

70

Treatment

Positive1X Yeast100 X Yeast

% S

alm

onel

la(+

)

Competitive ExlusionCompetitive Exlusion

1973 1973 -- Dr. Esko Nurmi and coDr. Esko Nurmi and co--workers in workers in Finland were the first to use the Finland were the first to use the competitive exclusion approach to control competitive exclusion approach to control salmonellae in broiler chicks.salmonellae in broiler chicks.

Likely Factors in the Exclusion ProcessLikely Factors in the Exclusion Process

Competition for receptor sitesCompetition for receptor sitesBacterial volatile fatty acidsBacterial volatile fatty acidsSubstrate competitionSubstrate competitionLow redox potentialLow redox potentialBacteriocinsBacteriocins

Summary of CESummary of CE1. Newly hatched chicks can be infected by 1. Newly hatched chicks can be infected by

a single cell of a single cell of Salmonella.Salmonella.2. Older birds are far more resistant to 2. Older birds are far more resistant to

colonization because of normal gut colonization because of normal gut microflora.microflora.

3. The 3. The introductionintroduction of flora from an adult of flora from an adult bird into newly hatched chicks speeds gut bird into newly hatched chicks speeds gut maturation and increases resistance to maturation and increases resistance to salmonella colonization.salmonella colonization.

Mucosal Starter Culture (MSC)Mucosal Starter Culture (MSC)

Mucosal Competitive Exclusion flora (MCE), Mucosal Competitive Exclusion flora (MCE), the basic material for the commercial the basic material for the commercial product Mucosal Starter Culture (MSC) is product Mucosal Starter Culture (MSC) is derived from the mucosal scrapings of ceca derived from the mucosal scrapings of ceca from healthy adult chickens which are then from healthy adult chickens which are then propagated under anaerobic conditions.propagated under anaerobic conditions.

Field Trial Testing of Mucosal Field Trial Testing of Mucosal Starter CultureStarter CultureTMTM

Results: Competitive ExclusionResults: Competitive Exclusion

Puerto RicoPuerto Rico On Farm:On Farm: 11 % to 2 %11 % to 2 %Processing Plant:Processing Plant: 41 % to 10 %41 % to 10 %

Georgia Georgia On Farm:On Farm: 2 % to 0 %2 % to 0 %Processing Plan:Processing Plan: 9 % to 4.5 %9 % to 4.5 %

TurkeyTurkey’’s At 6 Weeks:s At 6 Weeks: 40 % to 3 %40 % to 3 %

PrePre--Chill and PostChill and Post--Chill Chill Salmonella Salmonella DataDataPooled across all locations (3 trials)Pooled across all locations (3 trials)

P=0.02P=0.020+/1800+/1809+/1809+/180PostPost--ChillChill

P=0.05P=0.0512+/18012+/18023+/18023+/180PrePre--ChillChill

Chi SquareChi SquareMSC MSC

TreatedTreatedControlControlLocationLocation

Advantages of CE TreatmentAdvantages of CE Treatment

Easily appliedEasily appliedLow costLow costNonNon--specific protectionspecific protectionRapid host responseRapid host responseCompatible with other measuresCompatible with other measures

Antibiotic ResistanceAntibiotic ResistanceRisk / BenefitRisk / Benefit

Potential for direct transfer of resistance Potential for direct transfer of resistance from bacteria in the culturesfrom bacteria in the culturesPotential for genetic transfer between Potential for genetic transfer between bacteria in the CE mixturesbacteria in the CE mixturesCE has been shown to significantly CE has been shown to significantly reduce reduce C. perfringensC. perfringens induced necrotic induced necrotic enteritisenteritisLikely that the use of CE will allow the Likely that the use of CE will allow the significant reduction of subtherapeutic significant reduction of subtherapeutic antibiotics without loss of performanceantibiotics without loss of performance

Why not wide acceptance of Why not wide acceptance of Probiotics ?Probiotics ?

Many products contain Many products contain fewfew viable organismsviable organisms

Some products contain strains selected for growth in Some products contain strains selected for growth in

dairy products or at different temperaturesdairy products or at different temperatures

Antimicrobial or disinfectant interferenceAntimicrobial or disinfectant interference

Necessary to deliver high dosage (>10Necessary to deliver high dosage (>1066 CFU) of viable CFU) of viable

organisms in a short timeorganisms in a short time

How to Improve Probiotic How to Improve Probiotic Performance?Performance?

Genetic engineeringGenetic engineering•• GMO controversyGMO controversy

Continuous screening for probiotic Continuous screening for probiotic isolatesisolates•• Laborious but so far effectiveLaborious but so far effective

Better understanding of the GIT ecology:Better understanding of the GIT ecology:•• Improving conditions that favor probiotic Improving conditions that favor probiotic

bacteria activity / replication / establishmentbacteria activity / replication / establishment

•• Nutrition of bugs Nutrition of bugs →→ prebioticsprebiotics