probiotics-dfm-for-salmonella-control-in-poultry

FoodResearchInternational45(2012)628–633

ContentslistsavailableatScienceDirect

FoodResearchInternational

journalhomepage:http://wendang.chazidian.com/locate/foodres

Probiotics/directfedmicrobialsforSalmonellacontrolinpoultry

G.Telleza,?,C.Pixleyb,R.E.Wolfendenb,http://wendang.chazidian.comytona,1,B.M.Hargisa

ab

DepartmentofPoultryScience,UniversityofArkansas,Fayetteville,AR72701,USAPaci?cVetGroup-USA,Inc.Fayetteville,AR72704,USA

articleinfoabstract

Bacterialantimicrobialresistanceinboththemedicalandagricultural?eldshasbecomeaseriousproblemworldwide.Duringthelast15years,ourlaboratorieshaveworkedtowardtheidenti?cationofprobioticcandidatesforpoultrywhichcanactuallydisplaceSalmonellaandotherentericpathogenswhichhavecolonizedthegastrointestinaltractofchickensandturkeys,indicatingthatselectionoftherapeuticallyef?caciousprobioticcultureswithmarkedperformancebene?tsinpoultryispossible,andthatde?nedculturescansometimesprovideanattractivealternativetoconventionalantimicrobialtherapy.Ourstudieshavebeenfocusedonspeci?cpathogenreduction,performanceundercommercialconditions,andeffectsonbothidiopathicandde?nedenteritis.Wehavealsocon?rmedthatselectedheat-resistantspore-formingBacillusspeciescanmarkedlyreduceSalmonellaandClostridiumwhenadministeredinveryhighnumbers,andwehavedevelopedanovelandsimpletechniqueforobtainingculturedBacillussporecounts,providingacost-effectivefeed-stableinclusionincommercialpoultrydiets.Inordertoselectevenmoreeffectiveisolates,wearestillcurrentlyfocusedonthemechanisticactionoftheLactobacillusprobioticpreviouslydevelopedaswellasnewBacilluscandidates.Currentindicationsarethatmechanismofactioninvolvesrapidactivationofinnatehostimmuneresponses,providinganexcitingpossibilityforidenti?cationofvastlysuperiorandmorepotentprobiotics.Inthisreview,wesummarizethesafetyandef?cacyofindividualmonoculturesforprophylacticand/ortherapeuticef?cacyagainstSalmonellainfectionsunderbothlaboratoryand?eldconditionsaswellasthedevelopmentofanovel,cost-effective,feed-stabledirect-fedmicrobials(DFM)withpotentialforwidespreadutilizationandimprovedproduction,deliveryandclinicalef?cacyforanimaluse.

©2011ElsevierLtd.Allrightsreserved.

Articlehistory:

Received6January2011Accepted23March2011Keywords:Probiotic

DirectfedmicrobialsPoultrySalmonellaLactobacillusBacillus

1.Introduction

Salmonellaentericaserovarscontinuetobeamongthemostimportantfoodbornepathogensworldwideduetotheconsiderablehumanratesofillnessreported,thewidehostspeciesthatarecolonizedbymembersofthisremarkablepathogengenus,whichserveasvectorsandreservoirsforspreadingtheseagentstoanimalandhumanpopulations.Furthermore,thepublicconcernfortheappearanceofresistantstrainstomanyantibiotics,particularlyamongzoonoticpathogenssuchascommonSalmonellaisolates,isalsochallengingthepoultryindustryto?ndalternativemeansofcontrol(Boyle,Bishop,Grassl,&Finlay,2007).Forexample,inJanuary2006Europeimplementedacompletebanofgrowth-promotingantibioticsinanimalfeed(Anadon,Martinez-Larranaga,&AranzazuMartinez,2006).Thus,whileattemptingtodealwiththesehumanfood-bornepathogens,poultryproducersaresimultaneouslychal-lengedtoimproveproductioninthefaceofincreasingfeedcostswhileusingfewerantibioticsduetoincreasedrestrictionof

?Correspondingauthor.Fax:+14795758490.E-mailaddress:gtellez@uark.edu(G.Tellez).1

Currentaddress:ArgentinaVetancoS.A.Chile33(B1603CMA)VicenteLópez,BuenosAires,

Argentina.

0963-9969/$–seefrontmatter©2011ElsevierLtd.Allrightsreserved.doi:

10.1016/j.foodres.2011.03.047

antimicrobialusage.Theseinternalregulations,asinEurope,wereimplementedbecauseofexportmarketrestrictionsandconsumerorcustomerpreferencesinlocalmarkets.Forthesereasons,continuedresearchonsustainablealternativestoantibioticgrowth-promotersforanimalproductionsuchas:a)probioticsordirectfedmicrobials(DFM)consistingofliveordeadorganismsandspores(Patterson&Burkholder,2003);b)non-traditionalchemicals(Ko,Mendoncam,Ismail,&Ahn,2009);c)bacteriophages(AndreattiFilhoetal.,2007;Bielke,Higgins,Donoghue,Donoghue,&Hargis,2007;Higgins,etal.,2005;J.P.Higginsetal.,2008);andd)organicacidsandotherplantextractsandessentialoils(Aengwanich&Suttajit,2010;Allen-Hall,Arnason,Cano,&Lafrenie,2010;Bagchietal.,2000;Kubena,Byrd,Young,&Corrier,2001;Over,Hettiarachchy,Johnson,&Davis,2009;VanImmerseeletal.,2006)isincreasinglymoreimportant.Thesepotentialsolutionshaveemergedinthelastdecadesassomeofthetoolsthatcouldbepotentiallyusefulinthenearfutureforpathogencontrolandpoultryperformanceimprovement.

Probiosis,althoughnotanewconcept,hasonlyrecentlybeguntoreceiveanincreasinglevelofscienti?cinterest.Inagriculture,probiotics/DFMusedinanimalfeedarebecomingacceptedaspotentialalternativestoantibioticsforuseasgrowth-promoters,andinselectcases,forcontrolofspeci?centericpathogens(Anadón,RosaMartínez-Larrañaga,&AranzazuMartínez,2006;Boyleetal.,

G.Tellezetal./FoodResearchInternational45(2012)628–633629

2007;Cartman,LaRagione,&Woodward,2008;Vilaetal.,2009;Williams,Burdock,Jimenez,&Castillo,2009).Forthesereasonsthedevelopmentofnewandmoreeffectiveprobioticproductsthatcanbelicensedforanimalusecontinuestoreceiveconsiderableinterest(Hongetal.,2008a;Hong,Ducle,&Cutting,2005;Jadamus,Vahjen,&Simon,2001;Osipova,Makhailova,Sorokulova,Vasil'eva,&Gaiderov,2003;Williams,2007b;Wolken,Tramper,&vanderWerf,2003).

Currently,thereisnouniversalclassofprobioticbacteriumalthoughthemostcommontypesthathavebeenindisputablyeffectiveinvolvelacticacidbacteria.Thesebacteriaarefoundnormallyinthegastrointestinaltract(GIT)ofvertebratesandinvertebrates,andtheuseofsomelacticacidbacterialculturesisabletorestorethenaturalmicro?orawithinthegut(Shahani&Ayebo,http://wendang.chazidian.comcticacidbacteria(LAB)includethegeneraLactobacillus,Pediococcus,andothersthathavelongbeenassociatedwithhealthbene?tsandwhichhavebeenusedforfermentationofcertainfoods.Whilespeciationofmembersofthesegeneraisdif?cultandinconsistent,theseorganismsareuniformlysafeandnotassociatedwithdiseaseinhealthyanimalsorhumans(Tellez,Higgins,Donoghue,&Hargis,2006).

Asecondclassi?cationofprobioticculturesarethosemicroor-ganismsthatarenotnormallyfoundintheGIT(allochthonous?ora).Forexample,SaccharomycesboulardiihasbeenshowntobeeffectiveinpreventingtherecurrenceofClostridiumdif?cileinfections(Czerucka,Piche,&Rampal,2007)andsomecolibacillosisinhumans(Czerucka&Rampal,2002).Otherallochthonousprobioticmicrobesarethespore-formingbacteria,normallymembersofthegenusBacillus.Recentprogressinthisareafromourlaboratorywillbedescribedbelow.

2.Alacticacidbacteria-basedprobioticforSalmonellacontrolandperformanceinpoultry

Severalyearsago,ourlaboratoryevaluatedasimplemethodtoselectforindividualentericbacteriacapableofinhibitingSalmonellagrowthinvitroandtheabilityofselectedoxygentolerantbacteria,incombination,toprotectneonatalpoultsfromSalmonellainfectionfollowingchallenge.Concurrently,wealsoworkedtowardtheisolation,selection,furtherevaluationandcombinationofLABtocontroladditionalfoodbornepathogens(Tellezetal.,2006).Extensivelaboratoryand?eldresearchconductedwiththisde?nedLABculturehasdemonstratedaccelerateddevelopmentofnormalmicro?orainchickensandturkeys,providingincreasedresistancetoSalmonellasp.infections(Farnelletal.,2006;J.P.Higginsetal.,2008;Higginsetal.,2010;S.E.Higginsetal.,2008;Vicenteetal.,2008;Wolfenden,Pixley,Higgins,Higgins,etal.,2007;Wolfenden,Pixley,Vicente,Aviña,etal.,2007;Wolfenden,Vicente,Bielke,Pixley,etal.,2007;Wolfenden,Vicente,Higgins,Andreatti,etal.,2007).Publishedexperimentalandcommercialstudieshaveshownthattheseselectedprobioticorganismsareabletoreduceidiopathicdiarrheaincommercialturkeybroodinghouses(Higginsetal.,http://wendang.chazidian.comrgescalecommercialtrialshaveindicatedthatappropriateadministra-tionofthisprobioticmixturetoturkeysandchickensincreasedperformanceandreducedcostsofproduction(Torres-Rodriguezetal.,2007;Torres-Rodriguezetal.,2007;Vicenteetal.,2007;Vicente,Aviña,Torres-Rodriguez,Hargis,&Tellez,2007;Vicenteetal.,2007).

Thesedatahaveclearlydemonstratedthatselectionoftherapeuti-callyef?caciousprobioticcultureswithmarkedperformancebene?tsinpoultryispossible,andthatde?nedculturescansometimesprovideanattractivealternativetoconventionalantimicrobialtherapy.Thislacticacidbacteriabasedprobiotic(FloraMax-B11®)consistsof11bacteriarelatedtothegenusLactobacilluswhichwaslicensedtoacommercialcompany(Paci?cVetGroupUSAInc.,FayettevilleAR72703)andhasbecomeoneofthemoresuccessfulanteSalmonellastrategiesusedinthedrinkingwaterbythepoultryindustrytodate.Thebacterialcompositionislistedinthenextsection.

3.ThemechanismofactionofprobioticsagainstSalmonellaProbioticsarede?nedaslivemicroorganismsthat,whenadministeredinadequateamounts,conferahealthbene?tonthehost.Amongthemanybene?tsassociatedwiththeconsumptionofprobiotics,modulationoftheimmunesystemhasreceivedthemostattention(Borchers,Keen,&Gershwin,2002;Borchers,Selmi,Meyers,Keen,&Gershwin,2009).

Previously,itwasthoughtthatadministrationofbacteriasuchasprobioticstoneonatesdirectlyreducedinfectionbypathogensdueto‘competitiveexclusion’http://wendang.chazidian.competitiveexclusionwas?rstdescribedin1973byNurmiandRantalla(1973),citingthatbacteriacompetewitheachotherforspaceandnutrients.Theirdataindicatedthatearlyadministrationof‘good’bacteriapreventedinfectionbypathogens.SinceNurmiandRantalaproposedthatcompetitiveexclusioncouldbeusedasamethodtopreventSalmonellainfection,numerousresearchershavereportedtheabilityoflivebacterialcultures(Callawayetal.,2008;Corrieretal.,1998;Hollister,Corrier,Nisbet,&DeLoach,1999;Hume,Corrier,Nisbet,&DeLoach,1998;Nisbetetal.,1998;Wagner,Paine,&Cerniglia,2003)andprobioticorganisms(Higginsetal.,2007;Higginsetal.,2010;S.E.Higginsetal.,2008;Patterson&Burkholder,2003;Vicenteetal.,2008)toalsoreducecolonizationofopportunisticpathogensinthegastrointestinaltract.Yetourunderstandingofhowprobioticsmediatethesehealthbene?ts,speci?callyreductionofSalmonellainfection,isverylimited.

Balancedcolonicmicro?oraandimmunostimulationaremajorfunctionaleffectsattributedtotheconsumptionofprobiotics(Amit-Romach,Uni,&Reifen,2010;Boirivant,Amendola,&Butera,2008;Boirivant&Strober,2007;Flint,O'Toole,&Walker,2010;Flore,Francois,&Felicite,2010;Ibrahimetal.,2010;Klein,Sanders,Duong,&Young,2010;Nayak,2010).Manyprobioticeffectsaremediatedthroughimmuneregulation,particularlythroughbalancecontrolofproin?ammatoryandanti-in?ammatorycytokines(DiGiacinto,Marinaro,Sanchez,Strober,&Boirivant,1950;Foligneetal.,2010;Hacini-Rachineletal.,1950;Jobin,2010;Li,Xia,&Li,2009).However,severalanimalandhumanstudieshaveprovidedunequivocalevidencethatspeci?cstrainsofprobioticsareabletostimulateseveralaspectsofinnateimmunity(Amit-Romachetal.,2010;Boirivantetal.,2008;Boirivant&Strober,2007;Farnelletal.,2006;Romaninetal.,2010;Weissetal.,2010)aswellastoincreasehumoralimmunity(Fang,Elina,Heikki,&Seppo,2000;Galdeano,deLeblancAde,Carmuega,Weill,&Perdigon,2009;Leblanc,Fliss,&Matar,2004;Nermes,Kantele,Atosuo,Salminen,&Isolauri,2011).

UsingtheSalmonellachallengemodel,wehaveobservedanincreaseinSalmonellaenteritidis(SE)incidenceincecaltonsilsovertheinitial12hpost-treatment(Higginsetal.,2007;Higginsetal.,2010),whichindicatesthattheSEiscontinuingtocauseinfectiondespitetheoreticallycomingintocontactwiththeprobioticorganismswithinapproximately2h(neonatalchickgastrointestinaltransittime).Inarecentstudyconductedinourlaboratory,microarrayanalysisrevealedgeneexpressiondifferencesinbirdstreatedwithaLactobacillusbasedprobioticwhencomparedtosalinetreatedbirds.At12hpostprobiotictreatment,170genesweresigni?cantlydifferent(Pb0.05),butby24hposttreatment,thenumberofdifferentiallyregulatedgeneswas201.Pathwayanalysisrevealedthatatbothtimepoints,genesassociatedwiththeNFκBcomplexweresigni?cantlyregulated,aswellasgenesinvolvedinapoptosis.Probiotic-induceddifferentialregulationofthegenesGAS2andCYR61mayresultinincreasedapoptosisinthececaeofchicks.BecauseSalmonellaisanintracellularpathogen,wesuggestthatincreasedapoptosismaybeamechanismbywhichFloraMax-B11®reducesSalmonellainfection(Higginsetal.,2011).

4.Comparisonsbetweengenotypic16SrRNA,MIDI,andBiologidenti?cationsofFloraMax-B11®lacticacidbacteria

ItiswellrecognizedthatthespeciationofLABposesauniqueproblemformicrobialidenti?cation.Theidenti?cationtechniquesof

630G.Tellezetal./FoodResearchInternational45(2012)628–633

choiceformanyfacultativeanaerobesarebiochemicalanalyses,butthestandardidenti?cationsystemforlacticacidbacteriaiscellularfattyacidpro?ling.Nevertheless,thesephenotypicmethodscanyieldvariableresults.Genotypicmethodsthatrelyoncomparisonsof16SrRNAsequencesfromunknownbacteriaareprovingtobevaluableforuseinawiderangeofgeneraandarenotsensitivetovariablecultureconditions.Genotypic16SrRNAidenti?cationoforganismsfromprobioticculturesmaybemoreconsistentthanthecurrentstandardmicrobialtechniquesappliedseparatelytodifferentmicrobialgroups.However,thisapproachcomeswithitsownlimitationsandissues.Asidenti?cationisbasedonspeci?csequencehomologyascomparedwithaknowndatabaseofmicro?orapreviouslyidenti?edthroughconventionalmethodologies,thespeciationisdependentupontheclosestmatchwithwhatwaspreviouslyidenti?ed,correctlyorincorrectly,inthedatabase.Asdatabasesconstantlyexpandandchange,thesamesequencesubmissionovertimemaymatchothernameswithgreaterhomology.Thus,atthismomentitisnearlyimpossibletoreallyknowthespeciationofLABexceptunderspeci?cexampleswithveryhighlycharacterizedisolates.Infact,16SrRNAsequencingofisolatesfrominternationally-knownnamebrandsofcommercially-producedyogurtwithlivecultureshasconsistentlyresultedindatabasematcheswithLABspeciesthatarelabeledasotherspeciesontheyogurtlabels(unpublishedfromourlaboratory).Thus,while16sRNAsequencingcanpositivelyidentifyoneLABisolateasuniqueamongseveral,trueaccuracyofhomologycompar-isonsisasomewhatsubjectiveexercise.

Eventhoughtherearemanynewexperimentalmolecularidenti?cationtechniques,suchasmicroarrayhybridization,sequenceanalysisof16SrRNAisthepredominantmoleculartechnologypresentlyavailableformicrobialidenti?cationofthesecommensalmicroorganisms(Wagneretal.,2003),evenwiththeknownproblemofdatabaseaccuracyandconsistencyovertime.Thedetailedinformationneededtoidentifyeachspeciesrepresentedinacommercialprobioticproductcanonlybefullyobtainedfromthe16SrRNAatthelevelofthenucleotidesequence.Asanexample,ourlaboratoryhasdevisedanidenti?cationschemeusingtheMIDISystemIDfromtwodifferentprivatelaboratories(MicroTestLabInc.,Agawam,MA01001,USA;andMicrobialIDInc.,Newark,DE19713,USA)theBiologIDSystem(Biolog,Inc.,Hayward,CA94545,USA)andcomparedthoseresultswiththe16SrRNASequenceAnalyses(MicrobialIDInc.,Newark,DE19713,USA)foridenti?cationoftheindividualcomponentbacteriapresentinthecommercialprobioticFloraMax-B11®(Table1).TheresultsofthatstudyshowedthatthecomplexpopulationsofbacteriapresentinFloraMax-B11®arenoteasytoaccuratelyidentify,especiallywithphenotypictechniques.Conventionaltechnologiescandetecthumanpathogens,becausetheyarewell-establishedincomparativedatabases,butemergingandopportunisticpathogensarenot.Despitethefactthatuncertainty

existsbetweendifferentmethodsofidenti?cationofnon-pathogenicprobioticbacteria,identi?cationofknownpathogensismuchmoreconsistent.Therefore,theuseoffullyde?nedculturesforcompetitiveexclusionorprobioticusearestillinherentlysaferthanunde?nedculturesorthosewhereorganismsareidenti?edaftertheculturehasbeenproduced.

5.ABacillusspore-basedprobioticforSalmonellacontrolandperformanceenhancementinpoultry

InspiteofthesuccessshownbythedevelopmentoftheLABprobioticforuseincommercialpoultry(above),thereisstillanurgentneedforcommercialprobioticsthatareshelf-stable,cost-effectiveandfeed-stable(tolerancetoheatpelletizationprocess)toincreasecomplianceandwidespreadutilization.Amongthelargenumberofprobioticproductsinusetodaysomearebacterialsporeformers,http://wendang.chazidian.comedprimarilyintheirsporeform,some(thoughnotall)havebeenshowntopreventselectedgastrointestinaldisordersandthediversityofspeciesusedandtheirapplicationsareastonishing.WhilenotallBacillussporesarehighlyheattolerant,somespeci?cisolatesarethetoughestlifeformknownonearth(Vreeland,Rosenzweig,&Powers,2000)andcanbeusedunderextremeheatconditions.Severalstudieshaveshownthateitherlivevegetativecellsorendosporesofsomeisolatescanpreventcoloncarcinogenesis(Park,Jeon,Park,&Paik,2007)ordischargeantimicrobialsubstancesagainstGram-positivebacteria,suchasStaphylococcusaureus,Enterococcusfaecium,andC.dif?cile(O'Mahonyetal.,2001).Theseresultsprovidedevidenceofcolonizationandantimicrobialactivityofprobioticbacteria,thus,productscontainingBacillussporesareusedcommer-ciallyasprobiotics,andtheyofferpotentialadvantagesoverthemorecommonLABproductssincetheycanbeusedasdirect-fedmicrobials(Anadónetal.,2006;Barbosa,Serra,LaRagione,Woodward,&Henriques,2005;Ducle,Hong,Barbosa,Henriques,&Cutting,2004;Hongetal.,2005;Hongetal.,2008a;Hongetal.,2008b;McNulty,Boyle,Nichols,Clappison,&Davey,2007;Osipovaetal.,2003;Williams,2007a;Wolkenetal.,2003).Thereisscienti?cevidencesuggestingthatsomebutnotallisolatesofingestedBacillussubtilissporescan,infact,germinateinthesmallintestine(Casula&Cutting,2002;Casula&Cutting,2002;Ducle&Cutting,2003;Hoaetal.,2001).Together,thesestudiesnotonlyshowthatsporesarenottransientpassengersinthegut,buttheyhaveanintimateinteractionwiththehostcellsormicro?orathatcanenhancetheirpotentialprobioticeffect.Severalcommercialspore-formingBacilluscultureshavebeenshowntoreducefoodbornepathogens(Aureli,Fiore,Scalfaro,Casale,&Franciosa,2010).However,costissuesassociatedwithachievingnecessaryconcentrationsofsporesinfeedhavegreatlylimitedcommercialacceptanceintheanimalindustry(Hongetal.,2005).

Table1

ComparisonsbetweenMicroSeq,MIDI,andBiologidenti?cationsofFloraMax-B11®lacticacidbacteria.AdaptedfromTellezetal.,http://wendang.chazidian.comBID182427293637B4044464852

16SRNAsequencing(?rst500bp)MicrobialIDInc.

PediococcusparvulusWeissellaconfusaWeissellaconfusaPediococcusparvulusLactobacillussalivariusWeissellaconfusaWeissellaconfusa

WeissellaparamesenteroidesLactobacillussalivaruisLactobacillussalivariusPediococcusparvulus

MIDISYSTEMIDMicroTestLabInc.EnterococcuscecorumLactobacilluscaseiLactobacilluscasei

Lactobacillusdelbreuckii-bulgaricusLactobacilluscellobiosusPediococcusacidilacticiLactobacilluscasei

LactobacillusfermentumLactobacillushelveticusLactobacillushelveticusUnabletoidentify

MIDISYSTEMIDMicrobialIDInc.

LactobacillusgasseriLactobacilluscaseiLactobacilluscasei

Lactobacillusdelbreuckii-bulgaricusLactobacilluscaseiPediococcusruminisLactobacilluscellobiosusLactobacillusfermentum

LactobacillussanfranciscensisLactobacillusgasseriLactobacilluscellobiosus

BiologIDDept.ofPoultrySc.U.ofArkansas

Unabletoidentify

ClostridiumclostridiiformeWeissellaconfusa

LactobacillushamsteriWeissellaconfusaUnabletoidentify

WeissellaparamesenteroidesUnabletoidentify

LactobacillussalivaruisLactobacillussalivariusUnabletoidentify

G.Tellezetal./FoodResearchInternational45(2012)628–633631

Atthepresenttime,ourlaboratory'saimistodevelopanovel,cost-effective,feed-stableprobioticwithwidespreadutilizationandimproveprobioticproduction,deliveryandclinicalef?cacyforhumanandanimaluse.WehavedemonstratedthatoneB.subtilissporeisolatewasaseffectiveasourLAB-basedprobiotic(FloraMax-B11®)forSalmonellareduction(Shivaramaiahetal.,2011;Wolfendenetal.,2010),wasequaltobacitracinforpreventionofexperimentalnecroticenteritis,andwasabletomarkedlyreducenecroticenteritisissuesinlargescalefeedtrials(unpublishedexperiments).

Otherisolatesorcombinationsofisolateswithincreasedpotencyandef?cacymaybeidenti?edwithcontinuedresearch.SomeoftheseenvironmentalBacillusisolateshavebeenevaluatedinvitroforantimicrobialactivityagainstselectedbacterialpathogens,heatstability,andtheabilitytogrowtohighnumbers.Unpublishedexperimentalevaluationshavecon?rmedimprovedbodyweightgainaswellasSalmonellasp.orClostridiumperfringensreductionincommercialturkeyandbroileroperationswhencomparedwithmedicated(nitarsone)orcontrolnonmedicateddietsrespectively.Ourpreliminarydatasuggeststhattheseisolatescouldbeaneffectivealternativetoantibioticgrowth-promotersforcommercialpoultry.

Importantly,improvedef?ciencyofampli?cationandsporulationisabsolutelyessentialtogainwidespreadindustryacceptanceofafeed-basedprobioticforantemortemfood-bornepathogeninterven-tion;aswellascosteffectiveness.Recently,bothvegetativegrowthandsporulationratehavebeenoptimizedinourlaboratory,whichmayleadtonewef?cienciesforcommercialampli?cationandmanufactureofacost-effectiveproductatveryhighsporecounts(Wolfendenetal.,2010).Inordertoselectevenmoreeffectiveisolates,wearecurrentlyfocusedonthemechanisticactionofnewBacilluscandidates.PreliminarystudiesconductedinourlaboratoryindicatethatapotentialmechanisticactionofthesenewBacilluscandidatesatleastpartiallyinvolvesrapidactivationofinnatehostimmunemechanisms(systemorresponses)inchickensandturkeys(unpublisheddata).Thisdataprovidesanexcitingpossibilityforidenti?cationofvastlysuperiorandmorepotentprobioticsinthenearfuture.6.Conclusions

Theinterestindigestivephysiologyandtheroleofmicroorgan-ismshasgenerateddatawherebyhumanandanimalwellbeingcanbeenhancedandtheriskofdiseasereduced.Newmoleculartechniquesallowanaccurateassessmentofthe?oracompositionresultinginimprovedstrategiesforelucidatingmechanisms.Giventherecentinternationallegislationanddomesticconsumerpressurestowithdrawgrowth-promotingantibioticsandlimitantibioticsavailablefortreatmentofbacterialinfections,probioticscanofferalternativeoptions.Newadvancesintheapplicationofprobiotics,aredirectedtoproducesigni?cantchangesingutphysiologyandprovideevenhigherlevelsofhealthaswellasincreaseperformanceparametersinpoultry.

Metchnikofffoundedtheresearch?eldofprobiotics,aimedatmodulatingtheintestinalmicro?ora(Dobrogosz,Peacock,&Hassan,2010;Schmalstieg&Goldman,2010;Weissmann,2010).However,otherpartsofthebodycontainingendogenousmicro?oraorproblemsrelatingtotheimmunesystemmayalsobecandidatesforprobiotictherapy.Researchhasshownthatprobioticshavepotentialforhumanhealthissuessuchas:vaginalcandidiosis(Ehrstrometal.,2010;Ya,Reifer,&Miller,2010);dentalcaries(Chen&Wang,2010;Stamatova&Meurman,2009);allergies(Gourbeyre,Denery,&Bodinier,2011;Schiavietal.,2010b);autoimmunediseases(Lavasanietal.,2010;Tlaskalova-Hogenovaetal.,2011);urogenitalinfections(Pascual,Ruiz,Giordano,&Barberis,2010;Ruizetal.,2009);atopicdiseases(Hoang,Shaw,Pham,&Levine,2010;Nermesetal.,2011);rheumatoidarthritis(Leeetal.,2010;Mandel,Eichas,&Holmes,2010);andrespiratoryinfections(Harikrishnan,Balasundaram,&

Heo,2010;Silvestrietal.,2010).Currentresearchisstillheavilybiasedtowardgastrointestinalapplicationsforprobiotics,suchas:chronicconstipation(Bu,Chang,Ni,Chen,&Cheng,2007;Coccorulloetal.,2010);chronicdiarrhea(Preidisetal.,2011;Swidsinski,Loening-Baucke,Verstraelen,Osowska,&Doerffel,2008);in?ammatoryboweldisease(Ng,Chan,&Sung,2011;Vanderpool,Yan,&Polk,2008);irritablebowelsyndrome(Camilleri&Tack,2010;Enck,Klosterhalfen,&Martens,1946);andfoodallergy(Gourbeyreetal.,2011;Schiavietal.,2010a),butthepossibilitiesforimpactingmanyareasofhealtharenumerous.Muchresearchhasbeencompletedineffortstounderstandandapplythenaturalbene?tsofnon-pathogenicbacteria,butthereismuchstilltodo.References

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