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G Model VETPAR-7300; No. of Pages 9 ARTICLE IN PRESS Veterinary Parasitology xxx (2014) xxx–xxx Contents lists available at ScienceDirect Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar Effects of supplementation with dietary green tea polyphenols on parasite resistance and acute phase protein response to Haemonchus contortus infection in lambs Rong Zhen Zhong a,b , Hao Yang Li b , Hai Xia Sun a , Dao Wei Zhou a,∗ a b Northeast Institute of Geography and Agroecology
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  Pleasecitethisarticleinpressas:Zhong,R.Z.,etal.,Effectsofsupplementationwithdietarygreenteapolyphenolsonparasiteresistanceandacutephaseproteinresponseto Haemonchuscontortus infectioninlambs.Vet.Parasitol.(2014),http://dx.doi.org/10.1016/j.vetpar.2014.06.022 ARTICLE IN PRESS G Model VETPAR-7300;No.ofPages9VeterinaryParasitologyxxx(2014)xxx–xxx ContentslistsavailableatScienceDirect Veterinary   Parasitology  journalhomepage:www.elsevier.com/locate/vetpar Effects   of    supplementation   with   dietary   green   teapolyphenols   on   parasite   resistance   and   acute   phase   proteinresponse   to   Haemonchus   contortus   infection   in   lambs Rong   Zhen   Zhong a , b ,   Hao   Yang   Li b ,   Hai   Xia   Sun a ,   Dao   Wei   Zhou a , ∗ a NortheastInstituteofGeographyandAgroecology,ChineseAcademyofSciences,Changchun130102,Jilin,PRChina b CollegeofAnimalScienceandTechnology,JilinAgriculturalUniversity,Changchun130118,Jilin,PRChina a   r   t   i   c   l   e   i   n   f   o  Articlehistory: Received26October2013Receivedinrevisedform6June2014Accepted17June2014 Keywords:Haemonchuscontortus TeapolyphenolsResistanceAcutephaseproteinsLambs a   b   s   t   r   a   c   t The   objective   of    this   study   was   to   determine   the   effects   of    supplementation   with   dietarygreen   tea   polyphenols   (GTPs)   on   parasite   resistance   and   acute   phase   protein   (APP)   responseto Haemonchus   contortus   infection   in   lambs.   Thirty   male   Ujumqin   lambs   were   randomlyassigned   to   five   treatment   groups   for   an   8-week   feeding   period.   Treatments   included:   (1)uninfectedascontrol,   (2)infected   but   not   given   GTP   (INFGTP0)   and   (3)–(5)   infected   and   fed2,4,   or   6   g   GTP/kg   feed   (dry   matter   basis;   INFGTP2,   INFGTP4,   and   INFGTP6,   respectively).Fecal   and   blood   samples   were   collected   to   determine   fecal   egg   count   (FEC),   packed   cellvolume   (PCV),   and   APPconcentrations.   Live   weight   was   measured   once   every   2weeks.   Attheend   of    the   feeding   period,   lambs   were   slaughtered   to   determine   the   adult   H.   contor-tus   burden.   The   results   demonstrated   interaction   effects   between   treatment   and   samplingtime   onthe   average   dailygain   (ADG;   P    =0.0005),   FEC   ( P    <   0.0001),   PCV   ( P    =   0.0005),   andconcentrations   of    serum   amyloid   A(SAA),   haptoglobin   (Hp),   lipopolysaccharide   bindingprotein   (LBP),   and    1 -acid   glycoprotein   (  1 AGP)   ( P    <   0.0001).   From   days   0to   56,   the   ADGvalues   for   allinfected   lambs   were   lower   than   that   of    uninfected   lambs,   but   the   ADG   val-uesfor   all   GTP-fed   lambs   were   higher   thanthat   of    INFGTP0   lambs,   especially   from   days28   to   42.   The   FECs   of    allGTP-fed   lambs   were   higher   than   those   of    uninfected   lambs   butlower   thanthat   of    INFGTP0   lambs.   The   PCVsof    all   infected   lambs   were   lower   than   thoseof    uninfected   lambs,   but   PCV   increased   with   increasing   amounts   of    GTP   supplementation.Furthermore,   supplementation   with   different   concentrations   of    GTP   significantly   reducedthenumbers   of    adult   H.contortus ,   including   both   males   and   females   ( P    <   0.0001),   and   the   H.contortus   burden   in   INFGTP6   lambs   was   reduced   touninfected   levels.   Overall,   the   SAA,   Hp,LBP,   and    1 AGP   concentrations   of    all   infected   lambs   were   higher   than   those   of    uninfectedlambs   from   days   0to   56.   Two   peaks   in   expression   were   observed   from   days   0to   3   and   atday   28,   and   APP   concentrations   of    all   GTP-fed   lambs   were   lower   than   those   of    INFGTP0lambs,   except   for   SAA   in   INFGTP6   lambs.   Inconclusion,   quantitative   measurements   of    APPresponses   to   H.contortus   infection   provide   valuable   diagnostic   information   for   monitoringinfection   progression   and   treatment   responses   in   lambs.   An   appropriate   dose   of    dietary   GTP ∗ Correspondingauthor.Tel.:+8643185542231;fax:+8643185542206. E-mailaddress: zhoudaowei@neigae.ac.cn(D.W.Zhou).http://dx.doi.org/10.1016/j.vetpar.2014.06.0220304-4017/©2014ElsevierB.V.Allrightsreserved.  Pleasecitethisarticleinpressas:Zhong,R.Z.,etal.,Effectsofsupplementationwithdietarygreenteapolyphenolsonparasiteresistanceandacutephaseproteinresponseto Haemonchuscontortus infectioninlambs.Vet.Parasitol.(2014),http://dx.doi.org/10.1016/j.vetpar.2014.06.022 ARTICLE IN PRESS G Model VETPAR-7300;No.ofPages92 R.Z.Zhongetal./VeterinaryParasitologyxxx(2014)xxx–xxx supplementation   can   increase   host   resistance   byreducing   H.   contortus   burden   and   weightlossand   suppressing   blood   APP   expression.©2014   Elsevier   B.V.   All   rights   reserved. 1.Introduction Gastrointestinalnematodes(GINs)causeconsiderableeconomiclossesinsmallruminantproductionworldwide(ParkinsandHolmes,1989;MillerandHorohov,2006).The blood-feedingnematode Haemonchuscontortus isespe-ciallyharmful,causinganemiaandevendeathinhostanimals(Milleretal.,1998).InNorthernChina,approxi- mately90%ofsheepandgoatsareinfectedwith H.contortus (LiandLi,2006).TheindigenousUjumqinsheep,whichare famousforhavinghighqualitymeat,arelistedasanation-allyprotecteddomesticanimalbytheChinesegovernment.However,theyaresusceptibleto H.contortus infection,whichdecreasesproductivity(Naetal.,2010;Renetal.,2011).Currentprevalentparasitologicaldiagnosticapproachesaremostlybasedonthefecaleggcount(FEC),packedcellvolume(PCV)ofwholeblood,andFAMACHAscores(Niezenetal.,1998).TheseindirectindicatorsreflectGIN burden.DirectmeasurementsofGINinfectionreliesonnecropsyprocedurewhichisnotfeasibleinfarmcondi-tions.Therefore,ifmorepredictivebiomarkersassociatedwithimmuneresponsescanbeutilizedtomonitorGINbur-denanddiseaseprogression,moreaccuratetherapiescanbedevelopedtoimprovethehealthofhostanimals.Acutephaseproteins(APPs),includingserumamy-loidA(SAA),haptoglobin(Hp),lipopolysaccharidebindingprotein(LBP),and  1 -acidglycoprotein(  1 AGP),areagroupofbloodproteinsthatchangeinconcentrationwhenanimalsaresubjectedtoexternalorinternalchallenges,suchasinfection,inflammation,orstress(Gonzálezetal.,2008).ProductionofAPPsismediatedbypro-inflammatorycytokines,suchasinterleukin(IL)-1,IL-6,interferon(IFN)-  ,andtumornecrosisfactor(TNF)-  (Moshage,1997).SomestudieshaverevealedthatAPPsmay   beusefulaspotentialbiomarkersforevaluatinginfectionby Plasmod-iumchabaudi inmice(Taylor-Robinson,2000), Theileriaannulata incattle(Glassetal.,2003),and Trypanosomavivax insheep(SousaAlmeidaetal.,2012).However,therela- tionshipbetweenAPPinductionand H.contortus infectioninsheephasnotbeenwelldescribed.Anaccuratemethodforclinicaldiagnosisbasedonavarietyofparametersisultimatelyneededfortimelyadministrationofeffectivetherapies.Thecurrentstrategyofusinganthelminticstocontrol H.contortus isinjeopardybecauseofprevalentanthelminticresistance,drugresiduesinanimalproducts,andlossofproductivity(Waller,2004;Sykes,2010),whichhasstimulatedinterestinalterna-tiveparasitecontrolapproachesthatarelessreliantonchemotherapeutics.Severalstudieshavereportedthatgrazingontannin-containingplantscanreduceGINinfec-tioninsheepandgoats,withtanninsbeingtheeffectivebioactivecompounds(Niezenetal.,2002;Min   andHart,2003;Gujjaetal.,2013).Tanninsareaheterogeneousgroupofhighmolecularweightphenolicplantsecondarycompoundswiththecapacitytoformcomplexeswithpro-teins,polysaccharides,nucleicacids,etc.(Schofieldetal.,2001).Theproposedmechanismofnematodecontrolbytanninsinvolvesdirectbindingoftanninstonematodeparasiteproteins,whichsubsequentlyblocksthepara-site’sphysiologicalprocessesand/ormodifiesitsimmuneresponsestoeliminateinfectivelarvaeoradultworms(Hosteetal.,2006).Highlevelsofgalloylatedderivatives ofplantpolyphenolicextractgreatlycontributetotheanthelminticpotency(Azaizehetal.,2013).GreenteahasbeenthemostpopularbeverageinChinaforthousandsofyears(YangandWang,1993).Greentea containspolyphenoliccompounds,includingflavanols,fla-vandiols,flavonoids,andphenolicacids,whichaccountfor30%ofthedryweightofgreentealeaves.Mostof thepolyphenolsingreenteaarecatechins,including( − )-epicatechin(EC),( − )-epicatechin-3-gallate(ECG),( − )-epigallocatechin(EGC),and( − )-epigallocatechin-3-gallate(EGCG)(MukhtarandAhmad,2000).Greenteapolyphe- nols(GTPs)havebeenwidelyusedinanimalproductionduetotheirvariousbioactivities,suchasantioxidantproperties(Zhongetal.,2009).Basedonthechemi- calcompositionofGTPs,similartootherplant-derivedtannins,we   hypothesizedthatGTPshaveanthelminticbioactivitiescapableofreducingthe H.contortus burdeninsheep.Theobjectivesofthisstudywere:1)todeterminetheeffectsofdietaryGTPsupplementationonhostresistanceto H.contortus infectioninlambsand2)tostudytheAPPresponseto H.contortus infectioninlambs. 2.Materialsandmethods  2.1.Chemicalsandreagents Thegreentealeaves( Camelliasinensis L.)usedinthestudywas   suppliedbyHunanTeaGroupCo.,Ltd.,Hunan,China.GTPs(purityof98%)wereextractedfromgreentealeavesusinghighpressureliquidchromatog-raphy(ModelWaters600,WatersCo.,Milford,MA,USA)accordingtomethodof Pavetoetal.(2004).The extractedGTPscontainedtotaltannins(8.28%),steroids(4.12%),andflavonoids(85.2%),whichincluded(+)-catechinand( − )-catechin(DL-C)(1.21%),EC(5.07%),ECG(11.80%),EGC(0.47%),( − )-gallocatechingallate(GCG)(1.05%),EGCG(60%),andotherflavanols(5.6%).AllotherchemicalsusedinthestudywereobtainedfromSigmaChemicalCo.(St.Louis,MO,   USA)unlessotherwisestated.  2.2.Animalsandmanagement  TheuseoftheanimalsandtheexperimentalprocedurewereapprovedbytheAnimalCareCommittee,Instituteof   Pleasecitethisarticleinpressas:Zhong,R.Z.,etal.,Effectsofsupplementationwithdietarygreenteapolyphenolsonparasiteresistanceandacutephaseproteinresponseto Haemonchuscontortus infectioninlambs.Vet.Parasitol.(2014),http://dx.doi.org/10.1016/j.vetpar.2014.06.022 ARTICLE IN PRESS G Model VETPAR-7300;No.ofPages9 R.Z.Zhongetal./VeterinaryParasitologyxxx(2014)xxx–xxx 3  Table   1 Ingredientsandchemicalcompositionofthebasalexperimentaldiet.Ingredients(g/kgofDM) 1 Amount  Aneurolepidiumchinense 300Groundcorngrain475Solidsoybeanmeal(41%CP)150Molasses50Dicalciumphosphate 10Limestone5Tracemineralsaltandvitamins 2 10Chemicalcomposition( n =4)DM(g/kgoffeed)902.6 ± 10.65Crudeprotein(g/kgofDM)   176.8 ± 9.36Fat(g/kgofDM)   51.2 ± 1.36Neutraldetergentfiber(g/kgofDM)   584.6 ± 10.23Aciddetergentfiber(g/kgofDM)   358.4 ± 7.34Metabolizableenergy(MJ/kgofDM) 3 14.2 ± 0.13 1 DM=drymatter. 2 Mineralandvitaminsaltwas   purchasedfromContinentalGrainCorp.(Beijing,China)andcontained(perkg)24,000IUvitaminA,4800IUvita-min   E,120mg   Fe,and24mgCu. 3 MetabolizableenergywascalculatedaccordingtoNRC(1985). GeographyandAgroecology,ChineseAcademyofSciences, Jilin,China.ThirtymaleUjumqinlambswithanaverageageof 120 ± 10.3d,aninitialbodyweight(BW)of24.8 ± 2.4kg,andsimilargeneticbackgroundwererandomlyassignedtooneoffiveexperimentaldiets( n =6/group)forafeed-ingperiodof56daysinacompletelyrandomizeddesign.Althoughalllambswereseparatedfromadultnematode-infectedsheepandgivenGIN-freedietsfrombirth,alllambswerestilldewormedusingacombinationofmox-idectin(0.8mg/kgBW),levamisole(11.2mg/kgBW),andalbendazole(21.6mg/kgBW)   byinjectiontoeliminatethepresenceofanypossibleGINsuntilnofecaleggswerefoundusingtheMcMastertechnique(Staffordetal.,1994).All lambswerefedbasaldiets(Table1)formulatedaccording toNRC(1985).Treatmentsconsistedof:(1)uninfectedwith H.contortus ascontrol(UNINF),(2)infectedwith H.con-tortus butnotgivenGPTsupplementation(INFGTP0),(3)infectedwith H.contortus andgiven2gGPT/kgfeed(drymatter(DM)basisforall)supplementation(INFGTP2),(4)infectedwith H.contortus andgiven4gGPT/kgfeedsup-plementation(INFGTP4),and(5)infectedwith H.contortus andgiven6gGPT/kgfeedsupplementation(INFGTP6).Thethird-stage H.contortus larvaewerepreparedusingtheegg-hatchprocedureasdescribedpreviously(HansenandPerry,1990).Briefly,approximately5kgcrushedfecalpelletscollectedfrommaturesheepthatwerenaturallyinfectedwith H.contortus weremoistenedwithtapwaterandplacedinapanwithtwobeakerscontainingtapwatertomaintainhumidityinsidethepan.Thepanwas   keptatroomtemperaturefor10days.Thefecalsampleswerethenplacedinastrainerlinedwithfourlayersofcheese-cloth,andthestrainerwaskeptimmersedinapanof watertoallowthelarvaetosettletothebottom.Thecol-lectedlarvaewerewashedwithphosphate-bufferedsalinesolutionthreetimestoremovefecaldebris.Asampleoflar-vaecollectionwasthenstainedwithiodinetodeterminethespeciesbylightmicroscopy(Bausch&Lomb,Scien-tificOpticalProductsDiv.,Rochester,N.Y.,USA)accordingtotheparasitologicalmethoddescribedbyForeyt(1997).Theinfectivelarvaeof  H.contortus (third-stage)wereiden-tifiedaccordingtothefeatureoftailofsheathendinginafinewhip-likefilament(HansenandPerry,1990).Con- sequently,thepercentageof  H.contortus inthemixedlarvaewas   nolessthan95%,theotherspeciesoflarvawere Trichostrongyluscolubriformis and Oesophagostomumcolumbianum .Eachlambintheinfectedgroupswas   exper-imentallydrenchedwith5000third-stage H.contortus larvaeinonebatchbymouthatday0beforethemorningfeeding.Thefeedingexperimentlastedfor56days.Ateachfeed-ingday,GTPsupplementwasmixedwellwiththebasaldietaccordingtotherequiredratioofGTPtobasaldietbeforeeachfeedingtime.Alllambswerefedtwicedailyat06:00and18:00h,lambswerefed0.5kgfeed/head(DMbasis)eachfeedingtime,andtheamountoffeeddailywas   main-tainedfor56days.Theortsforeachlambwererecordeddailytocalculatefeedintake.Eachlambwas   assignedtoanindividualmetaboliccagewithaccesstofreshwater.  2.3.Samplingandanalyticalprocedures Thefeedsupplyandortsforeachlambwererecordeddailytocalculatefeedintake.Thendietsamplesweredriedat105 ◦ Cfor3htodetermineDMcontent.Thechemicalcompositionoftheortswasnotmeasured.Beforethemorningfeedingofdays0,14,28,42,and56oftheexperimentalperiod,alllambswereweighedandtheaveragedailygain(ADG)oflambswascalculatedbasedondifferencesinliveweightatspecificpointsintime.FecalsampleswerecollecteddirectlyfromtherectumofeachlambandprocessedimmediatelytodetermineFECusingtheMcMastertechniquereportedbyStaffordetal.(1994),withresultsexpressedaseggspergram(EPG)offeces.Beforethemorningfeedingofdays0,14,28,42,and56oftheexperimentalperiod,2-mlbloodsampleswerecollectedfromthejugularveinofeachlambbyvenipunc-tureintoheparin-containingvacutainers(BectonDickinon,VacutainerSystems,Rutherford,NJ,USA)todeterminePCVusingaUnicomicro-haematocritcentrifuge(182-E,Day-ton,NJ,USA)andamicro-capillaryreader(Damon/IECDivision).Beforethemorningfeedingofdays0,1,3,7,28,42,and56oftheexperimentalperiod,6-mlbloodsampleswerecollectedfromthejugularveinofeachlambintovacutainerswithoutanticoagulanttoharvestserumforAPPdetermination.TheconcentrationofSAAwasanalyzedusingacommerciallyavailablesandwichenzyme-linkedimmunosorbentassay(ELISA)kit(TrideltaDevelopment,Maynooth,Ireland)accordingtomethodof Piccioneetal.(2012).TheconcentrationofserumLBPwasquantifiedusingcommercialELISAkits(JianchengBiologyCo.,Nanjing,China).Briefly,theassayusedpurifiedgoatLBPantibodytocoatmicrotiterplatewells.LBPwasthenaddedtowells,whereitboundtoLBPantibody,whichwasthencomplexedwithhorseradishperoxidase(HRP)-labeledmouseanti-goatantibody.Thecolorchangewasmeasuredspectrophotometricallyatawavelengthof450.TheconcentrationsofserumHpand  1 AGPweremeasuredusingcommercialcolorimetrickits(JianchengBiologyCo.),whichfollowedasimilarprincipletothatoftheLBPassay.  Pleasecitethisarticleinpressas:Zhong,R.Z.,etal.,Effectsofsupplementationwithdietarygreenteapolyphenolsonparasiteresistanceandacutephaseproteinresponseto Haemonchuscontortus infectioninlambs.Vet.Parasitol.(2014),http://dx.doi.org/10.1016/j.vetpar.2014.06.022 ARTICLE IN PRESS G Model VETPAR-7300;No.ofPages94 R.Z.Zhongetal./VeterinaryParasitologyxxx(2014)xxx–xxx  Table2 Effectofsupplementationwithdifferentlevelsofdietarygreenteapolyphenolsonthetotal H.contortus counts( ± standarderror)recoveredfromabomasumof    lambsinfectedwith H.contortus andcontrollambs.Adult H.contortus Treatment 1 P  valueUNINFINFGTP0INFGTP2INFGTP4INFGTP6Total2 ± 1 d 1043 ± 42 a 421 ± 32 b 198 ± 15 c 83 ± 6 cd <0.0001Male   1 ± 1 c 410 ± 21 a 169 ± 15 b 68 ± 8 c 34 ± 3 c <0.0001Female   1 ± 1 d 633 ± 23 a 252 ± 22 b 130 ± 9 c 49 ± 5 cd <0.0001 a, b, c, d Meansvalueswithdifferentsuperscriptsinthesamerowwithintreatmentdiffer( P  <0.05). 1 UNINF=uninfected;INFGTP0=infectedandnotgivenGTP;INFGTP2=infectedandgiven2gGTP/kgfeed;INFGTP4=infectedandgiven4gGTP/kgfeed;INFGTP6=infectedandgiven6gGTP/kgfeed. Attheendofthestudy,alllambswereslaughteredbyexsanguinationwithpreviouselectrocution,andatnecropsy,theabomasumwasisolated,removed,andwashedinplasticbucketswith3Ltapwater.Aliquotsof 5%(150ml)   oftotaldigestawerecollectedin20duplicatesforeachlamb,and100ml   formalin(10%)wasaddedtoeachaliquot.Adult H.contortus from20duplicatedsamplesforeachlambwasrecovered,sexidentified,andcountedusingaphasecontrastmicroscope(LeicaInc.,Beijing,China),accordingtothemethodsof Minhoetal.(2008).  2.4.Statisticalanalyses AlldatawereanalyzedbySAS(2002)usingtheMIXED modelproceduredescribedbyLittelletal.(1996)withamodelconsistingofdietarytreatment,samplingtime,andtreatment × timeinteractionasfixedeffectswithanimalastherandomeffect.Themeasurementsobtainedfromeachlambatdifferentsamplingtimesweretreatedasrepeatedmeasures.TheEPGdatawerelog-transformed(ln(EPG+1))priortostatisticalanalyses.Meansweresep-aratedusingleastsquaresmeanandpresentedwiththestandarderrorofthemean.Statisticalsignificancewasdeclaredat P  ≤ 0.05. 3.Results  3.1.Averagedailygain TheADGdataforalllambsareshowninFig.1.Therewas nosignificantdifferenceofDMintakeamongfivegroups  Table3 Effectsofsupplementationwithdifferentlevelsofdietarygreenteapolyphenolsonserumacutephaseproteinconcentrationoflambsinfectedwith H.contortus andcontrollambs.AcutephaseproteinDayTreatment 1 SEM 2 P  valueUNINFINFGTP0INFGTP2INFGTP4INFGTP6TreatmentTimeIteractionsAmyloidA(mg/l) 01.131.141.030.910.99 6.437<0.001<0.001<0.0011   0.96383.481.485.876.331.3474.756.954.381.876.6186.278.765.991.6288.5413288.555.776.1420.98944.015.519.434.0561.6816.69.8318.636.3Haptoglobin(g/l) 00.0380.0520.0270.0620.045 0.125<0.001<0.001<0.0011   0.0430.5110.8280.7200.73330.0711.561.050.3090.79270.0450.9850.4760.1630.341280.1121.780.7500.3310.289420.0940.5730.1840.0860.137560.0511.050.5910.1920.170Lipopolysaccharidebindingprotein(g/l)01.511.811.701.561.71 0.369<0.001<0.001<0.0011   1.432.111.762.022.0431.629.157.416.542.9872.498.785.902.502.37282.7910.43.102.221.92421.204.373.271.463.87561.624.173.562.153.27  1 -Acidglycoprotein,g/L 00.2460.3150.2500.2700.303 0.056<0.001<0.001<0.0011   0.2850.3050.2790.3020.31630.2791.260.9890.5990.33070.3200.100.7790.3630.274280.4701.460.5940.3330.314420.2510.8420.4480.3990.359560.2740.9300.4190.2440.271 1 UNINF=uninfected;INFGTP0=infectedandnotgivenGTP;INFGTP2=infectedandgiven2gGTP/kgfeed;INFGTP4=infectedandgiven4gGTP/kgfeed;INFGTP6=infectedandgiven6gGTP/kgfeed. 2 SEM=standarderrorofthemean.
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