Research Update Methodology Improvements Methodology Improvements ... gene specific for the O104 serogroup.…

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  • ResearchUpdate

    Category:MethodologyImprovementsProjectTitle:ComparisonofnonO157ShigaToxinproducingE.coliDetectionSystemsMickBosilevac,USDA,ARS,USMeatAnimalResearchCenterObjective:ToidentifystrengthsandweaknessesofShigatoxinproducingEcolidetectionsystemsandkitsinasidebysidefashionThreecommercialShigatoxinproducingE.coli(STEC)detectiontestsandtwothirdpartylaboratorieswerecomparedtotheMLG(5B)andUSMARCmolecularmethodsforabilitytodetecttop6STEC(akaenterohemorrhagicE.coli[EHEC]).

    FSISMLG5BSTECDetection DuPontQualiconBAXSystemSTECSuitecommercialdetectiontest BioControlSystemsAssuranceGDSTopSTECcommercialdetectiontest PallGeneDiscSystemfornonO157STECcommercialdetectiontest IEHLaboratoriesthirdpartylab NeogenNeoSEEKthirdpartylab USMeatAnimalResearchCenterSTECdetectionandisolationprotocols

    Thestudydesignwasathreepartevaluationintwophases:

    Twoinoculationstudies Eachmethod/systemusedaccordingtoitsprotocol(medias,incubationtemperatures&times,

    material). Eachmethod/systemusedtotestthesameTSBenrichments(groundbeefvarying

    weight/volume/time). OnesetofRealWorldsamples

    Eachmethod/systemusedon500enrichmentsfromaregionalservicelab.Theresultsindicatedallmethods,usedasdirected,candetectlowlevelsofinoculatedtop6EHECthoughresultsvariedamongmethods(Table1).Onehundredsixtyof500sampleswerefoundtobepotentialpositivebyoneormoremethods,butonly2sampleswerepositivebyallmethods.ThisinformationallowsusersofSTECdetectionteststodrawcomparisonsandcriticallyconsiderthevalueofapotentialpositivewhenmultipleorganismsmaybepresent.Theresultsalsoprovideabaselineofwhatnumberofpotentialpositivesdifferentmethodsmaygive.

    2013BeefIndustrySafetySummit

  • ProjectTitle:EscherichiacoliO104inthefecesoffeedlotcattledonotharborgenetictraitsofthestrainresponsibleforthe2011GermanoutbreakT.G.Nagaraja,KansasStateUniversityObjective:TotestfeedlotcattlefeceswithamultiplexPCR(mPCR)designedtodetectserogroupO104andgenescharacteristicoftheSTECandEAECpathotypesandisolateandcharacterizeE.coliO104fromPCRpositivefecalsamples.EscherichiacoliO104:H4wasthecauseofalargefoodborneoutbreakofhemorrhagiccolitisandhemolyticuremicsyndromeinGermanyandotherEuropeancountriesin2011.TheserotypewasahybridofShigatoxinproducingE.coli(STEC)andenteroaggregativeE.coli(EAEC)pathotypes.BecausecattleareknownreservoirsofSTEC,theresearchersdesignedandvalidatedamPCRtodetectE.coliO104withSTECandorEAECtraitsbytargetingthefollowing8genes:stx1,stx2,terD(telluriteresistance),eae,wzxO104(O104specificOantigenflippase),fliCH4(H4specificflagella),ehxAandaggA(pilinsubunitofaggregativeadherencefimbriae).ThemPCRassayof248fecalsamples,collectedfrom8differentfeedlots,showedonlythreesamples(1.1%)werepositivebeforeenrichmentand51samples(20.6%)positiveafterenrichmentinECbrothforthewzxO104genespecificfortheO104serogroup.Noneofthe248fecalsamplescontainedtheaggA,agenethatischaracteristicoftheEAECpathotype(Table2).NosignificantassociationbetweenthepresenceofwzxO104andeitheroftheShigatoxingeneswasdetected.Onlyseven(13.7%)samplesyieldedpureculturesofserogroupO104andall7isolateswerenegativeforfliCH4andgenescharacteristicsofSTECandEAEC.TheseresultsshowthatcattlearenotalikelyreservoirforE.coliO104:H4withcharacteristicsofSTECandEAEC(Table2).

    Table2.PresenceofgenesthatencodeforEscherichiacoliO104serogroupspecifictraitsincattlefecalsamples(n=248)beforeandafterenrichmentinEscherichiacolibroth

    Genes(encodedproteinorfunction) Ampliconsize,Kb

    E.coliO104:H4(Germanstrain)

    No.ofsamplespositive(%)

    Beforeenrichment1

    Afterenrichment

    wzxO104(O104antigenflippase) 337 + 3(1.2) 51(20.6)fliCH4(H4flagellarantigen) 244 + 103(41.5) 214(86.3)stx1(Shigatoxin1) 655 37(14.) 144(58.1)stx2(Shigatoxin2) 477 + 100(40.3) 188(75.8)eae(Intimin) 375 92(37.1) 204(82.3)ehxA(enterohemolysin) 199 210(84.5) 243(97.8)terD(telluriteresistance) 434 + 119(48.0) 233(94.0)aggA(aggregativefimbrialadhesin) 151 + 0 0wzxO104+fliCH4 + 2(0.8) 47(20.0)wzxO104+fliCH4+stx1orstx2 + 2(0.8) 41(16.5)

    Category:PreharvestPathogenReductionProjectTitle:DirectFedMicrobialsasanAidintheControlofFoodbornePathogensinCattleLymphNodesandFecalSamplesMindyBrashears,TexasTechUniversityObjective:Todetermineifsupplementingdietswithahighdose(109/animal/day)ofLactobacillusacidophilusNP51(HNP51)willreduceSalmonellainlymphnodesatslaughteranddeterminereductionsinfecalpathogensforcattlesupplementedwithHNP51.

  • Thisprojectconsistedofbothcommercialandresearchfeedlotstudies.Thetreatmentsforbothstudieswerecontrols(i.e.,notfedHNP51)andanimalswhosedietsweresupplementedwith109/head/dayHNP51.Inthecommercialstudy,approximately1,800cattlewererandomizedintotwotreatmentswith12pens/treatmentand75head/pen.Intheresearchfeedlotstudy,112cattlewererandomizedto14pens/treatmentand4head/pen.Forthecommercialandresearchfeedlotstudies,Salmonellaprevalenceinthelymphnodes(Figure1)was25%(p

  • Table3.Modeladjusted,animallevel,fecalprevalenceestimatesforSTECOserogroupsandvirulencegenes(percentagesand95%confidenceintervals)bytypeofIMSbead.BoldtypeemphasizesserogroupprevalenceestimatesforthecorrespondingIMSbeadtype.

    Serogroupspecificandvirulencegenes O26IMSbeads O103IMSbeads O111IMSbeads

    O26 22.3(7.849.2) 3.0(0.5 16.0) 2.3(0.316.1)O103 3.1(1.18.6) 24.6(11.8 44.4) 17.7(6.440.4)O111 0.04(0.0195.8) 0.01(0.01 1.6) 0.01(0.0011.5)stx1 12.8(4.630.8) 8.8(4.0 18.0) 7.2(3.016.1)stx2 19.7(9.835.6) 18.4(9.5 32.8) 14.1(6.029.6)eae 8.8(3.520.5) 10.9(4.0 26.3) 9.2(3.522.2)ehxA 39.1(27.751.8) 45.7(32.4 59.6) 45.0(33.157.6)

    ThebaselineprevalenceestimatespresentedinthisstudyprovideinitialpointintimemeasuresofSTECfrequencyatthefeedlotlevel.Inaddition,thesedatacontributetoabetterunderstandingofthepotentialperformanceandlimitationsofSTECdiagnosticassaysandthedistributionofgenesindicatingthepotentialvirulenceofimportantserogroups.Category:PreandPostHarvestPathogenReductionSalmonellaProject Title:Do PreHarvest InterventionsApplied for Controlling E. coliO157:H7 in Feedlot CattleAffectFecalSheddingofNonO157E.coliorSalmonella?NataliaCernicchiaro,KansasStateUniversityObjective:Todeterminetheeffectsofcommerciallyavailablepreharvestinterventions(vaccineandadirectfedmicrobial)intendedtoreducefecalsheddingofE.coliO157infeedlotcattleonfecalprevalenceofsixnonO157ShigatoxinproducingE.coli(STEC)serogroupsandSalmonella,andwhetherthepresenceofnonO157STECandSalmonellaareassociatedwiththepresenceofO157withinfeedlotcattlefecalsamples.Morethan17,000cattlefedahighconcentratefinishingdietwith>25%distillersgrainsin40penswererandomizedtoreceiveone,bothorneither(control)ofthetwointerventions:asiderophorereceptorandporinproteinsbasedvaccine(E.coliSRPvaccine,Pfizer,NewYork,NY)andadirectfedmicrobialproduct(DFM;Bovamine,NutritionPhysiologyCo.,Guymon,OK).Cattleinthevaccinatedgroupsreceivedonedoseatenrollmentandagainthreeweekslater.TheDFMwasfed(106CFU/animal/day)throughoutthestudyperiod(mean=87days).DNAextractedfromfecalsampleswastestedfordetectionofO157andsixnonO157STECserogroups(O26,O45,O103,O111,O121,andO145)andfourmajorSTECvirulencegenes(stx1,stx2,eae,andehxA)byan11genemultiplexPCR(Baietal.,2012).Inaddition,aduplexrealtimePCRassay,basedoninvAandpagCgeneswasusedtodetectSalmonella.Theresultsshowed(Table4):

    Thevaccine,thedirectfedmicrobialorbothhadnostatisticallysignificanteffectonfecalprevalenceofnonO157STECorSalmonella.

    E.coliO157wasassociated(P

  • ThisinformationinthisstudyshowedthattwocommerciallyavailablepreharvestinterventionsdidnotreducefecalsheddingofnonO157orsalmonella.ProjectTitle:MethodsforControllingEscherichiacoliO157:H7andSalmonellaSurrogatesduringtheProductionofNonIntactBeefProductsLindseyMehall,TexasA&MUniversityObjective:ToevaluatethereductionandinternalizationofE.coliO157:H7andSalmonellasurrogatemicroorganismsbyapplyingfivedifferentantimicrobialtreatmentstosubprimalsurfacesbeforemarination.Striploinswereinoculatedwithoneoftwolevels(High[~107]andlow[~104])ofnonpathogenic,rifampicinresistantE.coliorganisms.Afteraging,eachstripwasassignedtooneoffiveantimicrobialtreatmentsorcontrol:2.5%Llacticacidand5.0%Llacticacidappliedat~53C,and1,050ppmacidifiedsodiumchlorite,205ppmperoxyaceticacid,andtapwaterappliedatroomtemperature(~25C).Treatedandcontrolloinswerevacuumtumbledwithacommercialmarinade,chilledfor24hours,vacuumpackaged,andagedfor7to24days.Sampleswerecollectedthroughouttheexperiment.Results:

    24hchillhadminimalimpactonsurfacesurrogatemicroorganismsforbothhighandlowinoculatedstrips.

    Thelogreductionsfollowingagingrangedfrom0.6to1.8log10CFU/cm2. Forbothhighandlowinoculatedstrips,the5%lacticacidtreatmenthadthegreatestlogreductionof

    surfacesurrogatemicroorganisms,whereaswaterhadtheleast. Followingantimicrobialtreatments,thelogreductionsofsurfacesurrogatemicroorganismsrangedfrom

    0.5to2.6log10CFU/cm2. Stripstreatedwithperoxyaceticacidhadthegreatestreductionofsurfacesurrogateorganismsinthe

    finished,marinatedproduct. Aftermarination,agreaterthanonelogreductionofsurfacesurrogatemicroorganismswasseenforall

    treatmentswiththeexceptionof2.5%lacticacid. Stripsreceivingwatertreatmentresultedingreaterinternalizationofsurrogatemicroorganismswhen

    comparedtothecontrol.

    Theresultsindicatethatlacticacid,acidifiedsodiumchlorite,andperoxyaceticacidsolutionsusedastreatmentscanminimizethenumberofsurfacepathogenscapableoftranslocatingorinternalizingduringtheproductionprocess.

  • ProjectTitle:VariationofSalmonellaPrevalenceinBovineLymphNodesamongSouthTexasFeedyardsAshleyN.Haneklaus,TexasA&MUniversityObjective:TodeterminetheimpactofcattlesourceonSalmonellaprevalenceinbovinelymphnodeslocatedinbeefproductsdestinedforfurtherprocessing.Atotalof307bovinelymphnodeswerecollectedinfourcollectiontripsoverathreemonthspanfromsevenfeedyardsselectedbasedonpredictedcontributiontotheharvestfacilitysSalmonellapositivetestresults(Table5).Salmonellaprevalenceinlymphnodeswasfoundtobe0%incattlesourcedfromoneofthesevenfeedyards.Lymphnodesfromcattlesourcedf