改性蒙脱石作为抗生素对断奶仔猪肠道菌群的影响
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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
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内容需要下载文档才能查看 内容需要下载文档才能查看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.
抗生素 肠道菌群
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
抗生素 肠道菌群
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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