改性蒙脱石作为抗生素对断奶仔猪肠道菌群的影响

抗生素 肠道菌群

AnimalFeedScienceandTechnology198(2014)257–262ContentslistsavailableatScienceDirect

AnimalFeedScienceandTechnology

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

Effectsofcetylpyridinium-montmorillonite,asalternativeto

antibiotic,onthegrowthperformance,intestinalmicro?ora

andmucosalarchitectureofweanedpigs

Y.L.Ke,L.F.Jiao,Z.H.Song,K.Xiao,http://wendang.chazidian.comi,J.J.Lu?,C.H.Hu??

AnimalScienceCollege,ZhejiangUniversity;TheKeyLaboratoryofMolecularAnimalNutrition,MinistryofEducation,

Hangzhou310058,ChinaarticleinfoabstractArticlehistory:

Received7July2014

Receivedinrevisedform8October2014

Accepted9October2014Keywords:

Antibiotic

Cetylpyridinium-montmorillonite

Intestinalmicro?ora

Mucosalarchitecture

WeanedpigsCetylpyridinium-montmorillonite(CP-Mt)wasusedasanalternativetochlortetracyclineongrowthperformance,intestinalmicro?oraandmorphologyinweanedpigs.Atotalof150piglets(Duroc×Landrace×Yorkshire,weanedat21±1dage)wereallottedto?vegroups:(1)control;(2)control+0.5g/kgCP-Mt;(3)control+1.0g/kgCP-Mt;(4)control+1.5g/kgCP-Mt;(5)control+75mg/kgchlortetracycline.Eachtreatmenthassixpenswith?vepigsperpen.Thefeedingtriallastedthreeweeks.TheresultsshowedthatincrementalCP-Mtinclusioninthedietincreasedaveragedailygain(linearP=0.013;quadraticP=0.034),decreasedEscherichiacoliandStreptococcussuisinjejunalcontentslinearly(P=0.004andP=0.006)andquadratically(P=0.014andP=0.009),decreasedE.coliandStreptococcussuisincolonlinearly(P=0.005andP=0.001)andquadratically(P=0.007andP=0.004),increased

villusheight(linearP<0.001;quadraticP=0.001)andtheratioofvillusheightandcrypt

depth(linearP<0.001;quadraticP=0.001),reducedplasmadiamineoxidase(DAO)(linear

P=0.000;quadraticP=0.001),andincreasedjejunalmucosaDAO(linearP=0.006;quadratic

P=0.002).Thepigletsfed1.0g/kgand1.5g/kgCP-Mtdidnotdifferfromthosefedchlorte-

tracycline(P>0.05)ingrowthperformance,intestinalmicro?oraandmucosalarchitecture.

TheresultsindicatedthatCP-Mtcouldbeagoodcandidateasanantibioticalternativein

weanedpigs.

©2014ElsevierB.V.Allrightsreserved.1.Introduction

Early-weanedpigletscommonlyencounterlowfeedintake,bodyweightlossandpost-weaningdiarrhea(Huetal.,2013b;McLambetal.,2013;Smithetal.,2010).Thereisalargenumberofevidenceshowingthatweaningdisturbsintestinalmicrobiotaandimpairsintestinalmorphology(Huetal.,2012b,2013c;Jiaoetal.,2014).Antibioticiswidelyusedtoreduceentericinfectionsandtheoccurrenceofpathogensabletoadheretointestinalmucosa(KilandStein,2010).TheincreaseduseofantibioticshasgivenrisetoaconcernofthedevelopmentofresistantpathogenicbacterialstrainsandresidualAbbreviations:ADFI,averagedailyfeedintake;ADG,averagedailygain;SEM,standarderrorofthemean;CP-Mt,cetylpyridinium-montmorillonite;DAO,diamineoxidase.

?Correspondingauthor.??Correspondingauthor.Tel.:+8657186961553;fax:+8657186961553.

E-mailaddresses:jjlu@http://wendang.chazidian.com(J.J.Lu),chhu@http://wendang.chazidian.com(C.H.Hu).

http://wendang.chazidian.com/10.1016/j.anifeedsci.2014.10.010

0377-8401/©2014ElsevierB.V.Allrightsreserved.

抗生素 肠道菌群

258Y.L.Keetal./AnimalFeedScienceandTechnology198(2014)257–262

contaminationofthefoodchainwithantibiotic(Vondruskovaetal.,2010).In2006,theuseofantibioticsasgrowthpromoterswasforbiddenintheEU.

Specialattentionhasbeenpaidtonaturalclayminerals,whichwerenotonlyusedasantibioticalternatives(Philip,2013;Songetal.,2012),butalsousedascarriersofantibacterialagent(HuandXia,2006;Özdemiretal.,2010).Montmorillonite(MMT),analuminosilicateclay,hasbeengenerallyusedasanantimicrobialcarrierduetoitslargespeci?csurfaceareaandhighcationexchangecapacity.TherecentstudiesaremainlyfocusedonimmobilizationofinorganicantimicrobialcationssuchasCu2+andZnOontomontmorillonite(Huetal.,2012a,2013a;Songetal.,2013b).

Immobilizationoforganiccationswithantibacterialactivityhassomeadvantagescomparedtothatofinorganiccations(Herreraetal.,2000).Cetylpyridiniumchlorideasaquaternaryammoniumsaltissuchanagentusedinantisepticsolutions,and?ndsawiderangeofantibacterialapplicationsinoralrinsesandthroatlozenges,inthemeatindustryforthedecon-taminationofcarcasses,andinthefurtherprocessingofpoultry,pork,andbeefforthecontrolofmicrobialgrowth(Özdemiretal.,2013).Previousinvestigationsdemonstratedthatcetylpyridinium-montmorillonite(CP-Mt)exhibitedstrongantibac-terialactivityforpathogenicbacteria,suchasSalmonellaenteritidisandStaphylococcusaureus(Herreraetal.,2000;Özdemiretal.,2013).

However,therearenodataontheeffectsofCP-Mtinvivo.Inthisexperiment,wehypothesizedthatCP-Mtcouldbeusedasalternativetochlortetracyclineongrowthperformance,intestinalmicro?oraandmucosalarchitectureinweanedpigs.

2.Materialsandmethods

2.1.Materials

Themontmorillonite(ChifengWHTBMiningCo.,LTD,Chifeng,China)contentwas99.0%.Thecationexchangecapacity(CEC)was1.30moL(+)/kgmontmorillonite.Thecetylpyridinium-montmorillonite(CP-Mt)wassynthesizedaccordingtothemethodofHerreraetal.(2000)andÖzdemiretal.(2013).Montmorillonitewashydratedwithwatertowhichcetylpyridiniumchloridewasaddedatanamountof0.5timestheCECofthemontmorillonite.Theresultingmixturewasthenagitatedandallowedtoexchangefor24h.TheCP-Mtwasthenseparatedbycentrifugationandwashedunderagitationfor24hwith100mLdeionizedwater.Thewashedmaterialwasdriedat60?Cfor24handground.

2.2.Experimentaldesignandsamplescollection

AllprocedureswereapprovedbytheUniversityofZhejiangInstitutionalAnimalCareandUseCommittee.Atotalof150weaningpigs(Duroc×Landrace×Yorkshire),withanaverageinitialweightof6.2kgweanedat21±1d,wereallocatedtothe?vegroupsforthreeweeks,eachofwhichwasreplicatedsixtimeswith?vepigsperreplicate.The?vegroupswere:

(1)control;(2)control+0.5g/kgCP-Mt;(3)control+1.0g/kgCP-Mt;(4)control+1.5g/kgCP-Mt;(5)control+75mg/kgchlortetracycline.DietswereformulatedtomeetorexceedrequirementssuggestedbytheNationalResearchCouncil(1998)(Table1).Allpigsweregivenadlibitumaccesstofeedandwater.Averagedailygain(ADG),averagedailyfeedintake(ADFI),andgain/feedratioweremeasured.

Afterthefeedingtrial,sixpigletsfromeachtreatment(onepigperpen)werekilledbasedonaveragebodyweight.Bloodsampleswerecollectedfromtheanteriorvenacavaintotubescontainingsodiumheparinandmixedimmediatelytoavoidcoagulation.Plasmawasobtainedaftercentrifugationat3000×gfor15minat4?Candthenstoredat?80?Cuntilanalysis.Specimensofthedistaljejunumandproximalcolonwere?xedin10%formalinformorphologymeasurements.Theintestinalcontentsfromdistaljejunumandproximalcolonwerecollectedformicrobiotaanalysis.Mucosalsamplesfromthedistaljejunumwerecollected,rapidlyfrozeninliquidnitrogenandstoredat?80?Cforfurtheranalysis.

2.3.Sampleanalysis

16SribosomalRNA-basedmethodswereusedfortheabundancesofEscherichiacoliandStreptococcussuisasdescribedbyHuijsdensetal.(2002)andSuetal.(2008).TotalDNAwasextractedfromintestinalcontentsusingaTIANampStoolDNAKit(TiangenBiotech),(TaKaRaBiotechnology,Dalian,China)accordingtothemanufacturer’sinstructions.Real-timePCRwasperformedon7500realtimePCRsystems(AppliedBiosystems,FosterCity,USA)usingFastSYBR®GreenMasterMixSYBRGreenchemistry(AppliedBiosystems,FosterCity,USA).ForE.colitheforwardprimersequencewas5??-CATGCCGCGTGTATGAAGAA-3??,thereverseprimersequencewas5??-CGGGTAACGTCAATGAGCAAA-3??(Huijsdensetal.,2002).ForStreptococcussuistheforwardprimersequencewas5??-CAGTATTTACCGCATGGTAGATAT-3??,thereverseprimersequencewas5??-GTAAGATACCGTCAAGTGAGAA-3??(Maroisetal.,2004).

Threecross-sectionsforeachjejunalsamplewerestainedwithhematoxylinandeosinusingstandardparaf?nembeddingprocedures.Cryptdepthandvillusheightweremeasuredinatleast10well-orientedcrypt-villusunitsusingimageanalysis(LeicaImagingSystemsLtd.,Cambridge,England)andaveragedforeachsample.

Diamineoxidase(DAO;EC1.4.3.6)activitiesinplasmaandintestinalmucosaweremeasuredusinganenzymaticspec-trophotometricassayasdescribedbyHuetal.(2012a).DAOactivitiesinplasmaandintestinalmucosawereexpressedasU/mLandU/mgprotein,respectively.

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Y.L.Keetal./AnimalFeedScienceandTechnology198(2014)257–262

259

Table1

Ingredientandchemicalcompositionofthebasaldietasfedbasis.

Ingredients(g/kg)

Corn

Soybeanmeal

Extrudedfull-fatsoybeanFishmeal

Spray-driedplasmaproteinDriedwheySoybeanoil

DicalciumphosphateLimestone

Sodiumchloridel-LysineHCldl-Methionine

Vitamin-mineralpremixa

5601601203925501511.55310.51014.35224.514.64.39.47.5

Analyzedcomposition(g/kg)Digestibleenergyb(MJ/kg)CrudeproteinLysine

MethionineCalcium

Totalphosphorus

Providedperkilogramofdiet:vitaminA,5500IU;vitaminD3,500IU;vitaminE,40IU;ribo?avin,5.0mg;vitaminB12,0.03mg;pyridoxine,3.0mg;vitaminK3,1.0mg;biotin,0.10mg;thiamine,2.0mg;niacin,30mg;pantothenicacid(calciumpantothenate),20mg;folicacid,0.6mg;choline(cholinechloride),800mg;Zn(ZnSO4),100mg;Fe(FeSO4),125mg;Cu(CuSO4·5H2O),16mg;Mn(MnSO4·H2O),15mg;I(KI),0.2mg;Se(Na2SeO3),0.3mg.b

DigestibleenergywascalculatedfromdataprovidedbyFeedDatabaseinChina(2012).

a

2.4.Statisticalanalysis

StatisticalanalysiswasperformedbytheSPSS9.0statisticalpackage(SPSSInc.,Chicago,IL).AlltreatmentswerecomparedbyANOVAusingTukey’scorrectionformultiplemeancomparison.PolynomialcontrastswerealsousedtoassessthelinearandquadraticeffectsofdietaryCP-Mtinclusionlevel.Alphalevelfordeterminationofsigni?cancewas0.05.

3.Results

3.1.Growthperformance

GrowthperformanceofpigletsispresentedinTable2.IncrementalCP-MtinclusioninthedietincreasedADG(linearP=0.013;quadraticP=0.034).Ascomparedwithcontrol,supplementationwithchlortetracyclineimproved(P<0.05)ADG.ThegrowthperformanceoffedwithCP-Mtdidnotdifferfromthosefedwithchlortetracycline(P>0.05).

3.2.Intestinalmicrobiota

Intestinalmicro?oraofpigsispresentedinTable3.IncrementalCP-MtinclusioninthedietdecreasedE.coliandStrepto-coccussuisinjejunalcontentslinearly(P=0.004andP=0.006)andquadratically(P=0.014andP=0.009).IncrementalCP-MtinclusioninthedietdecreasedE.coliandStreptococcussuisincolonlinearly(P=0.005andP=0.001)andquadratically(P=0.007andP=0.004).TheabundancesofE.coliandStreptococcussuisofpigsfedCP-Mtdietdidnotdifferfromthosefedchlortetracycline(P>0.05).

Table2

Effectofcetylpyridinium-montmorilloniteongrowthperformance.

Control

CP-Mta(g/kg)

Chlortetracycline

SEMbPcLinear

0.5

1.0

1.5

Quadratic0.0340.6810.292

ADGd(g)ADFIe(g)Gain/feed

266y3630.73

275xy3620.76

286x3700.77

285x3690.77

290x3720.78

5.97.40.02

0.0130.3750.159

CP-Mt,cetylpyridinium-montmorillonite.Standarderrorofmeans.c

EffectofCP-Mtadditionbypolynomialcontrasts.d

ADG,averagedailygain.e

ADFI,averagedailyfeedintake.xy

Meanswithinarowwithdifferentlettersdiffersigni?cantly(P<0.05).

a

b

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260

Y.L.Keetal./AnimalFeedScienceandTechnology198(2014)257–262

Table3

Effectofcetylpyridinium-montmorilloniteonintestinalmicrobiotaaandmucosalarchitecture.

Control

CP-Mtb(g/kg)

Chlortetracycline

SEMcPdLinear

0.5

1.06.51y5.22y7.65y6.73y767x3202.40x1.79y0.28x

1.5

Quadratic

JejunumE.coli

JejunumStreptococcussuisColonE.coli

ColonStreptococcussuisVillusheight(?m)Cryptdepth(?m)

Villusheight:cryptdepthPlasmaDAO(U/mL)

JejunalmucosaDAO(U/mgprotein)

7.20x6.20x8.42x7.67x629y3501.80y2.55x0.19y

6.83xy5.58xy7.93xy7.10xy680y3382.01y2.13xy0.24xy

6.42y5.26y7.69y6.57y763x3232.37x1.63y0.26x

6.30y5.19y7.62y6.48y758x3292.30x1.73y0.26x

0.180.230.170.2325.512.60.070.160.02

0.0040.0060.0050.001<0.0010.0820.001<0.0010.006

0.0140.0090.0070.0040.0010.1960.0010.0010.002

xy

Meanswithinarowwithdifferentlettersdiffersigni?cantly(P<0.05).

Bacterialnumbersareexpressedaslog10(16SrRNAgenecopiesg?1wetweight).b

CP-Mt,cetylpyridinium-montmorillonite.c

Standarderrorofthemean,n=6.d

EffectofCP-Mtadditionbypolynomialcontrasts.

a

3.3.Intestinalmorphology

Table3showsjejunalmorphologyofpigs.IncrementalCP-Mtinclusioninthedietincreasedvillusheight(linearP<0.001;quadraticP=0.001),andtheratioofvillusheightandcryptdepth(linearP=0.001;quadraticP=0.001).Thepigletsfed1.0and1.5g/kgCP-MtdidnotdifferinjejunalmorphologyfromthosefedChlortetracycline(P>0.05).

3.4.PlasmaandintestinalmucosaDAO

PlasmaandjejunalmucosaDAOactivityispresentedinTable3.IncrementalCP-MtinclusioninthedietreducedplasmaDAO(linearP<0.001;quadraticP=0.001),andincreasedjejunalmucosaDAO(linearP=0.006;quadraticP=0.002).TheDAOactivityofpigsfedCP-Mtdietdidnotdifferfromthosefedchlortetracyclinediet(P>0.05).

4.Discussion

Inrecentyears,claymineral-basedantibacterialcomplexeshavebeenpreparedbyaseriesofprocessesbetweentheclaymineralsandantibacterialsubstances(HuandXia,2006;Özdemiretal.,2010).Montmorillonitesareasubsetofalu-minosilicateclayshavinga2:1layerstructure.Withinthelayersoftheseclays,substitutionofothermetalionsforsiliconoraluminumcanoccurresultinginanetnegativechargeonthesurfaceoftheclayplatelet.Thisnegativechargeisoff-setbyhydratedcations,thepredominantonesbeingNa+andCa2+(Herreraetal.,2000).Theseinterlaminarcationscanbeexchangedwithantibacterialcations,suchasCu2+andZn2+(Malachovaetal.,2011).Thecopper-exchangedmontmorilloniteexhibitedstrongantibacterialactivity(HuandXia,2006).Invivostudiesshowedthatcopper-exchangedmontmorilloniteandZnO-montmorillonitehybridalleviateddiarrhea(Huetal.,2012b),modi?edintestinalmicro?oraandmucosalbarrierintegrityofweanedpigs(Huetal.,2012a,2013a;Songetal.,2013b).However,thereisincreasingevidencethatantimicrobialresistancegenesarepositivelycorrelatedwithCuandzincconcentrationsintheenvironment(ChoudhuryandSrivastava,2001;Marketal.,2000).Manystudiesshowedthatheavymetalsincludingcopperandzinc,usedinanimalfarmingmightpromotethespreadofantibioticresistanceviaco-selectionand/orothermechanisms(Baker-Austinetal.,2006;Holzeletal.,2012).Linaetal.(2011)foundthatzincresistanceandtheczrCgenearewidespreadamongCC398MRSAisolates.Thissug-gestedthattheuseofzincinfeedmighthavecontributedtotheemergenceofantimicrobialresistance.ThusCu-exchangedmontmorilloniteandZnO–montmorillonitehybridmightalsocontributetoantimicrobialresistance.

Clay-basedorganicanti-bacterialagentswerepreparedbyareactionbetweenclayandorganiccationswithantibacterialactivity,andhavesomeadvantagescomparedtothatofinorganiccations(Herreraetal.,2000),suchasincreasedstrengthandheatresistanceandincreasedbiodegradability(SinhaRayandOkamoto,2003)andlongerdurationofantibacterialef?cacy(Özdemiretal.,2013).Cetylpyridiniumchloride(CP)isamemberofafamilyofquaternaryammoniumsaltswhichincludeapositivelychargedhydrophilicammoniummoietyandhydrophobicalkylchains.TheantimicrobialactivityofCPisduetoitsabilitytoalterthepermeabilityofcellularmembranesallowingintracellularionsandlowmolecular-weightmetabolitestodiffuseout(Herreraetal.,2000).Depletionoftheseintracellularconstituentscausesthebacterialcelltoconsumeavailableenergystoresinanattempttorestorehomeostasis,resultingincellulardeath(Herreraetal.,2000).Previousinvestigationsdemonstratedthatcetylpyridinium-montmorillonite(CP-Mt)exhibitedstrongantibacterialactivityforpathogenicbacteriainvitro,suchasSalmonellaenteritidisandStaphylococcusaureus(Herreraetal.,2000;Özdemiretal.,2013).Thepresentstudyshowedforthe?rsttimethatsupplementationwithCP-MtdecreasedE.coliandStreptococcussuisinintestinalcontents.ExchangingmontmorillonitewithCPproducesamaterialthathasanetpositivechargeandismorehydrophobicthantheparentclay(Herreraetal.,2000;Özdemiretal.,2013).TheCP-exchangedclayenhancedhydrophobicityandaf?nityforpathogenicbacteria.Twopossiblemechanismsfortheantibacterialactivitywereproposed.

抗生素 肠道菌群

Y.L.Keetal./AnimalFeedScienceandTechnology198(2014)257–262261

Onemodelinvolvedtheadsorptionofthebacteriaandimmobilizationonthesurfaceoftheorganoclay.Alternatively,CPcoulddisassociatefromtheclaysurfaceanddirectlyexertsitsantimicrobialeffectonthebacteria(Herreraetal.,2000;Özdemiretal.,2013).

Thestructureoftheintestinalmucosacanrevealsomeinformationonguthealth.Stressorscanleadrelativelyquicklytochangesintheintestinalmucosa(Songetal.,2013a).Weaningisassociatedwithvillusatrophyandcrypthyperplasia(Huetal.,2013b;Wijttenetal.,2011).Ashorteningofthevillusdecreasesthesurfaceareafornutrientabsorption.Thecryptistheareawherestemcellsdividetopermittherenewalofthevillus,andalargecryptindicatesfasttissueturnoverandahighdemandfornewtissue.Inthepresentstudy,increaseinvillusheightandvillusheighttocryptdepthratioatthesmallintestinalmucosaofthepigssupplementedwithCP-Mtwasobserved.Suchimprovedintestinalmucosalmorphologymayberelatedtothechangesinintestinalmicrobiota,andinthepresentstudynumbersofE.coliandStreptococcussuislowered.

Theintegratedintestinalbarrieristopreventthetranslocationofintestinalbacteria,andtheenteringoftoxicorallergenicsubstancesfromthegutintothebody(Wijttenetal.,2011).Injuredintestinalbarrierincreasedtheepithelialpermeability(Wijttenetal.,2011).TheintestinalmucosaDAOactivityiscloselyrelatedwithvillimaturationandproteinsynthesisofmucosalcells.ThenormalhealthymucosapreventstheDAOintheintestinallumenfromenteringthecirculation.TheDAOisnormallypresentinverysmallamountsinthecirculation(WolvekampanddeBruin,1994).Whentheintestinalbarrierfunctioniscompromised,increasedmucosalpermeabilityallowsmoreDAOtoentertheperipheralcirculation.PlasmaDAOhasbeenproposedascirculatingmarkersformonitoringtheextentofintestinalbarrierinjury(Fukudomeetal.,2014).Alargebodyofevidencehasdemonstratedthattheearlyweaningprocessimpairedintestinalbarrierfunctionofpiglets(Huetal.,2013b;McLambetal.,2013;Smithetal.,2010).InthepresentstudyshowedthatCP-MtsupplementationincreasedintestinalmucosaDAOwhilereducedplasmaDAO,indicatingthebarrierfunctionwereimprovedbyCP-Mtfeeding.

5.Conclusion

Supplementationwith1.0g/kgand1.5g/kgCP-Mtinweaneddietswasaseffectiveaschlortetracyclineforimprovinggrowthperformance,mucosalarchitectureandmodifyingintestinalmicro?ora.TheCP-Mtcouldbeagoodcandidateasanantibioticalternativeinweanedpigs.

Con?ictofinterest

Nonedeclared.

Acknowledgment

ThisresearchwassupportedbytheSpecialFundforAgro-scienti?cResearchinthePublicInterest(No.201403047).References

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