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CapaCity Building for SurveillanCe and prevention of BSe and other ZoonotiC diSeaSeS

course manual

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DIAGNOSTIC TECHNIQUES FORTRANSMISSIBLE SPONGIFORMENCEPHALOPATHIES

food and agriCulture organiZation of the united nationSRome, 2007

Catherine BotteronMarcus DoherrDagmar Heim

Elizabeth MumfordTorsten Seuberlich

The designations employed and the presentation of material in this information product do not imply the expression of any opinion whatsoever on the part of the Food and Agriculture Organization of the United Nations concerning the legal or development status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries.

All rights reserved. Reproduction and dissemination of material in this information product for educational or other non-commercial purposes are authorized without any prior written permission from the copyright holders provided the source is fully acknowledged. Reproduction of material in this information product for resale or other commercial purposes is prohibited without written permission of the copyright holders. Applications for such permission should be addressed to the Chief, Electronic Policy and Support Branch, Communication Publishing Division, FAO, Viale delle Terme di Caracalla, 00153 Rome, Italy or by e-mail to [email protected]

© FAO 2007

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ContentS

Foreword v

Courseobjectives vii

introduCtion to tranSmiSSiBle Spongiform enCephalopathieS

1. Transmissible spongiform encephalopathies 1

2. Bovine spongiform encephalopathy 3

3. Measures for control and prevention 5

4. Clinical signs 10

5. Surveillance systems 11

6. Risk assessment 13

7. References 14

laBoratory-BaSed SurveillanCe and epidemiology

1. Monitoring and surveillance systems 19

2. Diagnostic test characteristics 23

3. Sampling issues 28

4. Measures of association 31

5. References 35

6. Suggested software 35

7. Solutions for exercises 35

BioSafety

1. General concepts 37

2. Biosafety for TSEs 38

3. References 40

Bovine Spongiform enCephalopathieS diagnoStiC methodology

1. BSE diagnosis at the TSE Reference Laboratory, Berne 41

2. Sample collection 41

3. Neuropathology and immunohistochemistry 43

4. Rapid BSE tests 48

5. References 50

iv

appendiX 1

Course contributors and staff 51

appendiX 2

Related background reading and Web links 55

appendiX 3

Glossary of technical terms and acronyms 61

appendiX 4

Project summary 69

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foreword

Tosupportcountrieswitheconomiesintransitionanddevelopingcountriesinthecon-trol and prevention of bovine spongiform encephalopathy (BSE), the project CapacityBuilding for Surveillance and Prevention of BSE and Other Zoonotic Diseases, is theresult of collaboration between the Food and Agriculture Organization of the UnitedNations(FAO),SafeFoodSolutionsInc.(SAFOSO,Switzerland)andnationalveterinaryofficesinpartnercountries,andfundedbytheGovernmentofSwitzerland.

Theaimoftheproject istobuildcapacity,establishpreventivemeasuresandana-lyserisksforBSE.PartnercountriesarethusenabledtodecreasetheirBSErisktoanacceptablelevelordemonstratethattheirBSEriskisnegligible,andtherebyfacilitateregional and international trade under the Agreement on the Application of SanitaryandPhytosanitaryMeasures(SPSAgreement)oftheWorldTradeOrganization(WTO).Abriefprojectsummaryisincludedasanappendixtothiscoursemanual.

Activitiesoftheproject:• Thespecificneedsofpartnercountriesareassessed.• Fourcomprehensivecoursesto“trainthetrainers”areprovidedtoselectedpar-

ticipantstoimproveunderstandingoftheepidemiologyofandrelevantriskfac-torsforBSEandtransmissiblespongiformencephalopathy(TSE)andtodevelopspecificknowledgeandskillsforimplementingappropriatecontrols.

• Inathirdstep, in-countrycoursesareheldbytrainednationalpersonnel inthelocallanguageandaresupportedbyanexperttrainer.

FAOhasthemandatetoraiselevelsofnutritionandstandardsofliving,toimproveagriculturalproductivityandthelivelihoodsofruralpopulations.Surveillanceandcon-trolofdiseasesofveterinarypublichealthimportancearecontributionstothisobjec-tive.SAFOSO,aprivateconsultingfirmbasedinSwitzerland,isprovidingthetechnicalexpertiseforthisproject.

ThismanualisasupplementtothetrainingcourseDiagnostictechniquesfortrans-missible spongiform encephalopathies, which is given within the framework of theproject.Thispracticalcourseistargetedatveterinarydiagnosticianswhowillcontributeto thedevelopmentand implementationof thenationalBSEsurveillanceandcontrolprogramme,andtotheBSEriskassessmentforthepartnercountries.

Theinformationincludedinthemanualisnotintendedtobecompleteortostandonitsown.Forfurtherreading,specificreferencesareincludedattheendofthechapters.General background material and Web links, and a glossary of terms and frequentlyusedacronyms,areincludedasappendices.

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ThepreparationofthismanualwasacollaborativeeffortofthetrainersoftheDiag-nostic techniques for transmissible spongiform encephalopathies course offered inSwitzerlandandtheprojectstaff.Thecontentofthemanualreflectstheexpertiseandexperienceoftheseindividuals.FAOandSAFOSOaregratefultotheprofessionalspre-paringthemanualandtotheGovernmentofSwitzerlandforfundingthispublic–privatepartnershipprojectinsupportofsaferanimalproductionandtrade.

Samuel C. Jutzi ulrich Kihm Director Director FAOAnimalProductionandHealthDivision SafeFoodSolutions Rome,Italy Berne,Switzerland

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CourSe oBJeCtiveS

UponcompletionofthelecturesandexercisesofthecourseonDiagnostictechniquesfor transmissible spongiform encephalopathies, of the project Capacity Building forSurveillance and Prevention of BSE and Other Zoonotic Diseases, the participantsshould:

• understandbasic informationonBSEandTSEs, includingtransmission,patho-genesis,riskvariablesandepidemiology;

• understandtheconceptsoftestingforBSE,includinglimitations;• beabletocollectappropriatebrainsamplescorrectlyfromcattleheads;• Beabletopreparebrainsamplescorrectlyforhistopathology,immunohistochem-

istryandrapidtests;• beabletorunrapidtests;• beabletodiagnoseBSEcorrectlyusingimmunohistochemistryandrapidtests.

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introduCtion to tranSmiSSiBle Spongiform enCephalopathieS

1. tranSmiSSiBle Spongiform enCephalopathieSTransmissible spongiform encephalopathies (TSE) are a class of neurodegenerativediseasesofhumansandanimalscharacterizedbyspongiformdegenerationofthebrainandtheassociatedneurologicalsigns.TSEsareslowlydevelopinganduniformlyfatal.

Diseasesincludekuru,Gerstmann-Sträussler-ScheinkersyndromeandCreutzfeldt-Jakobdisease(allinhumans),scrapie(insheepandgoats),felinespongiformencepha-lopathy (FSE; in cats), bovine spongiform encephalopathy (BSE; in cattle), chronicwasting disease (CWD; in cervids) and transmissible mink encephalopathy (TME; inmink).MostoftheseTSEshadalreadybeenreportedbeforethefirstdetectionofBSE(Figure1)(Lasmezas,2003).

FIGURE 1

year in which the various tSes were first reported

TheTSEwith the longesthistory is scrapie,whichwas recognizedasadiseaseofsheepinGreatBritainandothercountriesofwesternEuropemorethan250yearsago(DetwilerandBaylis,2003).Scrapiehasbeenreportedinmostsheep-raisingcountriesthroughouttheworldwithfewnotableexceptions(e.g.Australia,NewZealand).

Transmissibleminkencephalopathy(TME)wasfirstdescribedin1947.Itisararedis-easeoffarmedminkandhasbeenrecordedincountriesincludingtheUnitedStatesofAmerica(USA),Canada,Finland,GermanyandtheRussianFederation.ContaminatedfeedissuspectedtobethemainsourceofTMEinfection.

Chronicwastingdisease(CWD)incaptiveandfree-roamingNorthAmericandeerandelkwasfirstdescribedinthe1960s.Initially,caseswereonlyreportedincaptivedeerandelkinColorado(USA),butCWDincaptiveand/orfreeroamingdeer,elkandmoosehasnowbeenreportedinseveralotherstatesintheUSAandinareasofCanada.TheoriginofCWDisstillunknown.

1700 1750 1800 1850 1900 1950 2000

Variant Creutzfeldt-Jakob disease

Feline spongiform encephalopathy

Bovine spongiform encephalopathy

Chronic wasting disease

Transmissible mink encephalopathy

Gerstmann-Sträussler-Scheinker syndrome

Creutzfeldt-Jakob disease

Kuru

Scrapie

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Scrapie, kuru, Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker syn-drome,TME,andCWDarebelievedtobedistinctfromBSE.However,straintypinghasindicatedthatsomeotherTSEsarecausedby thesamestrainof theTSEagent thatcausesBSEincattle.OnlyfouryearsaftertheinitialBSEcaseshadbeendiagnosedincattleintheUnitedKingdomofGreatBritainandNothernIreland(UK),BSEindomes-ticcats(felinespongiformencephalopathy/[FSE])wasfirstreported.Almostalloftheapproximately100FSEcasesdiagnosedworldwideoccurredintheUK.Themostwidelyacceptedhypothesis is that theaffecteddomesticcatswereexposedtoBSE infectiv-ity throughcontaminatedcommercialcat feedor freshslaughteroffal thatcontainedbrainorspinalcordfrombovineBSEcases.SeverallargecatskeptinzooswerealsodiagnosedwithFSE.Theseincludedcheetahs,lions,ocelots,pumasandtigers.AllofthelargecatsthatwerediagnosedwithFSEoutsidetheUKoriginatedfromUKzoos.It issuspected that these largecatsacquired the infectionbybeing fedcarcassesofBSE-infectedcattle.

NotlongafterBSEwasdiagnosedincattle,sporadiccasesofBSEinexoticruminants(kudus,elands,Arabianoryx,ankolecows,nyala,gemsbockandbison)werediagnosedinBritishzoos.OnezebuinaSwisszoowasalsoBSEpositive.Inthemajorityofthesecases,exposuretoanimalfeedproducedwithanimalprotein(andthereforepotentiallycontainingBSEinfectivity)waseitherdocumentedorcouldnotbeexcluded.

Moreover,therehaslongbeenconcernthatsheepandgoatscouldhavebeenexposedtoBSE,becauseithasbeenexperimentallydemonstratedthatBSEcanbeorallytrans-mittedtosmallruminants(SchreuderandSomerville,2003). In2005,thefirstcaseofBSEinagoatwasconfirmedinFrance(Eloitetal.,2005),thoughtherehavebeennocon-firmedBSEcasesinsheeptodate.ItisdifficulttodistinguishbetweenscrapieandBSEinsheep,asdifferentiationiscurrentlynotpossiblebyclinicalorpathologicalmeans.

SeveralTSEshavebeenreportedtooccurinhumans,includingtwoformsofCreut-zfeldt-Jakobdisease(sporadicCJDandvariantCJD[vCJD]),Kuru,Gerstmann-Sträus-sler-Scheinkersyndrome,aswellas fatal familial insomnia.Of these,onlyvCJDhasbeenassociatedwithBSE.SporadicCJDwasfirstidentifiedin1920asanencephalopa-thyoccurringalmostexclusivelyinelderlypatientsworldwide.TheincidenceofsporadicCJD isapproximately0.3–1.3casespermillion individualsperyear,and issimilar inmostcountries.Thedurationofthediseaseisapproximatelysixmonths.Approximately80-89%ofCJDcasesarebelievedtobesporadic,10%arefamilial(aresultofaheritablemutationinthePrPgene),andtheremainderarebelievedtobeiatrogenic.

VariantCJDwasfirstreportedinMarch1996intheUK(Willetal.,1996).IncontrasttosporadicCJD,patientsareyoung(averageage29years)andthedurationofthediseaseis longer(average22months).Epidemiologically, little isknownaboutvCJD. Insomecasesthediseasewasseeningeographicalclusters,andthereareindicationsthatspe-cialconsumptionpatternsmayhaveplayedarole.Geneticfactorsmayalsoplayaroleininfection,aspatientswithclinicaldiseasehavebeenhomozygousformethionineatcodon129oftheprionproteingene.InEurope,thisgenotypeaccountsforapproximately30%ofthepopulation.

The expected course of the vCJD epidemic is difficult to predict, since importantvariablessuchashumanexposurerate,theinfectiousdose,theincubationperiodandhumansusceptibilityarelargelyunknown.Thepredictions initiallyrangedfromafewhundredtoafewmillionexpectedcases.However,thelowerpredictionsaremoreprob-ablebasedonthecurrentincidenceofvCJDcases(Figure2).

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ThelinkbetweenBSEandvCJDiscommonlyaccepted.Initially,thetemporospatialassociationoftheoutbreakssuggestedacausalrelationship.Experimentally,inocula-tionoftheBSEagentintothebrainsofmonkeysproducesfloridplaqueshistologicallyidenticaltothosefoundinthebrainsofvCJDpatients.Inaddition,theagentsassociatedwithBSEandvCJDaresimilar,bothbyglycotyping(evaluatingtheglycosylationpattern)andbystraintyping,whereastheprionsassociatedwithotherTSEs(suchassporadicCJD,scrapieandCWD)aredifferent.

2. Bovine Spongiform enCephalopathy2.1. origin and spreadBSEwasfirstdiagnosedincattleintheUKin1986(Wellsetal.,1987).Extensiveepide-miologicalstudieshavetracedthecauseofBSEtoanimalfeedcontaininginadequatelytreated ruminant meat and bone meal (MBM) (Wilesmith et al., 1988). Although ele-mentsofthescenarioarestilldisputed(e.g.originoftheagent;Wilesmithetal.,1991;Princeetal.,2003;SSC,2001a), itappears likely thatchanges inUKrenderingproc-essesaround1980allowedtheetiologicalagenttosurviverendering,contaminatetheMBMandinfectcattle.Someoftheseinfectedcattlewouldhavebeenslaughteredatanolderage,andthereforewouldhavebeenapproachingtheendoftheBSEincuba-tionperiod.Potentially, theyhadnoclinical signsor thesignsweresubtleandwentunrecognized,thoughthecattlewouldhaveharbouredinfectivitylevelssimilartothoseseen inclinicalBSEcases.Thewasteby-products from thesecarcasseswould thenhavebeenrecycledthroughtherenderingplants,increasingthecirculatinglevelofthepathogen(whichbynowwouldhavebecomewelladaptedtocattle)intheMBM,thuscausingtheBSEepidemic.

In1989thefirstcasesoutsidetheUK,intheFalklandIslandsandOman,wereidenti-fiedinlivecattlethathadbeenimportedfromtheUK.In1989Irelandreportedthefirstnon-imported(“native”or“indigenous”)caseoutsidetheUK,andin1990Switzerlandreportedthefirst indigenouscaseontheEuropeancontinent. Indigenouscaseswere

FIGURE 2

number of vCJd cases in the uK over time

1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006

30

25

20

15

10

5

0

3

10

10

18

15

28

20 17

18

9

31

Source:DepartmentofHealth,UK(2006)

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thenreportedinmanycountriesthroughoutEurope.In2001,JapanreportedthefirstindigenouscaseoutsideEurope,andthiscasehasbeenfollowedbyindigenouscasesinIsraelandNorthAmerica.1

2.2. epidemiologyCattletestingpositiveforBSEhaverangedfrom20monthsto19yearsofage,althoughmostofthecasesarebetweenfourandsixyearsofage.Abreedorgeneticpredisposi-tionhasnotbeenfound.MostcasesofBSEhavecomefromdairyherds,likelyduetodifferencesinfeedingsystemswhencomparedtobeefcattle.Additionally,beefcattlearetypicallyyoungeratthetimeofslaughter.Becausetheaverageincubationperiodisfourtosevenyears,infectedbeefcattlewillgenerallynotlivelongenoughtodevelopclinicalsigns.

Thereisnoexperimentalorepidemiologicalevidencefordirecthorizontaltransmis-sionofBSE,andthereisstillcontroversyregardingthepotentialforverticaltransmis-sion.Noinfectivityhasthusfarbeenfoundinmilk(TAFS,2007;SSC,2001b),ova,semenor embryos from infected cattle (SSC 2002a, 2001c; Wrathall, 1997; Wrathall et al.,2002).SomeoffspringofBSEcasesintheUKwerealsoinfected,andacohortstudyofUKcattleconcludedthatverticaltransmissioncouldnotbeexcluded.However,theroleofvariationingeneticsusceptibilityorothermechanismsinthisconclusionisunclear,andnooffspringofBSEcaseshavebeenreportedwithBSEoutside theUK. Ifsomeamountofmaternaltransmissiondoesoccur,it isclearlynotenoughtomaintaintheepidemic,evenwithintheUK.

2.3. pathogenesis Intheearly1990s,infectivitystudiesofBSEincattlewereongoing.Atthattime,experi-mental inoculation of tissues from BSE-infected cattle into mice had only identifiedinfectivity inbraintissue.Therefore,definitionofspecifiedriskmaterials(SRM;thosetissuesmostlikelytobeinfective)wasbasedonscrapieinfectivitystudies.Scrapierep-licatesprimarilyinthelymphoreticularsystem,andscrapieinfectivityhasbeenfoundinnumerouslymphnodes,tonsils,spleen,lymphoidtissueassociatedwiththeintestinaltractandplacenta.Duringthelaterpreclinicalphase,infectivityisfoundinthecentralnervoussystem(CNS).Inaddition,scrapieinfectivityhasbeendetectedinthepituitaryandadrenalglands,bonemarrow,pancreas,thymus,liverandperipheralnerves(SSC,2002b).

The first resultsofBSEpathogenesisstudies, inwhichcalveswere intracerebrallyinoculatedwithtissuefromBSEfieldcasesandfromcattleexperimentallyinfectedbytheoral route,becameavailable in themid-1990s (Wellsetal.,1996;1998). Incattleexperimentally infectedby theoralroute,BSE infectivityhasbeen found in thedistalileumatspecificintervalsduringtheincubationperiod,startingsixmonthsafterexpo-sure(Wellsetal.,1994).Furthermore,CNS,dorsalrootgangliaandtrigeminalgangliawerefoundtobeinfectiveshortlybeforetheonsetofclinicalsigns.Recently,lowlevelsof infectivity early in the incubation period have been detected in the palatine tonsil.Inonestudy,sternalbonemarrowcollectedduringtheclinicalphaseofdiseasewasinfective;however,thisresulthasnotbeenreproduced(thereforeitmaypossiblyhavebeenduetocrosscontamination)(Wellsetal.,1999;Wells,2003).

1 CurrentthroughJanuary2007.

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2.4. tSe agentsAlthough some controversy still exists regarding the nature of the BSE agent, mostresearchersagreethataresistantprionproteinisthecauseofthedisease.Researchhasshowntheagenttobehighlyresistanttoprocessesthatdestroyothercategoriesofinfectiousagents,suchasbacteriaandviruses,andnonucleicacidhasbeenidenti-fied.

Ineukaryoticspecies,mostcellscontainanormalprionprotein,termedPrPC(super-script “C” for “cellular”).Thisprotein isnormallydegradablebyproteases.TSEsarethoughttobecausedbyanabnormal,infectiousformofPrPC,inwhichthestericconfor-mationhasbeenmodifiedandwhichishighlyresistanttoproteinasedegradation.ThisinfectiousformismostcommonlytermedPrPSc(initiallyfor“scrapie”),butmayalsobereferredtoasPrPBSEorPrPRes(fortheportionthatis“resistant”toaspecificproteinase,proteinaseK).BecauseprionproteinisverycloselyrelatedtothenormalcellularPrPCprotein,itdoesnotinducetheproductionofantibodiesininfectedanimals.

TheroleofPrPCinnormalanimalsisstillunderdiscussion.GeneticallymodifiedmicelackingthegeneforPrPC(andexpressingnoPrPC)canbeexperimentallyproduced,butthesemicehavenoobviousphysiologicalchangesthatcanbeattributedtolackingtheprotein.Theycannot,however,beinfectedexperimentallywithTSEagents.

3. meaSureS for Control and prevention3.1. aims of measuresTheultimateaimsofBSEcontrolandpreventionprogrammesaretoreduceexposureriskbothtocattleandtohumans(Figure3).Twolevelsofmeasuresmustthereforebeconsidered:

• thosethatblockthecycleofamplificationinthefeedchain;• thosethatpreventinfectivematerialfromenteringhumanfood.

Owing to the prolonged incubation period, it may be more than five years betweeneffective enforcement of measures and a detectable decrease in the number of BSEcases,i.e.beforetheeffectofthemeasuresisseen.Thisintervalmaybeevenlongerifthemeasuresarenotenforcedeffectively,asisusuallythecaseforsometimeafterimplementation.

FIGURE 3

reducing BSe exposure risk to both cattle and humans

Blockamplification

EradicateBSE

Prevent the BSE agent from entering human food

Prevent animal exposure

Prevent human exposure

Recycling andamplification cycle in cattle

MeasuresIntermediate

goalsUltimate

aims

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RiskmanagementforBSEisnotgloballyharmonized.InEurope,thememberstatesoftheEuropeanUnion(EU)havecommonrulesfortheimplementationofmeasures,andothercountriesinEuropeandcountrieswantingtojointheEUareadaptingtheirmeasures accordingly. However, the implementation of these measures still variesconsiderablyfromonecountrytoanother.

3.2. measures to protect animal healthfeed bansRecognitionofMBMasasourceofinfectionledtobansonfeedingMBMtoruminantsinordertobreakthecycleofcattlere-infection(DEFRA,2004a;EC,2004;HeimandKihm,1999).Implementationofa“feedban”maymeandifferentthingsindifferentcountries.FeedscontainingMBMofruminantormammalianoriginmightbebanned,orthebanmight includeallanimalproteins (i.e.mammalianMBM, fishmealandpoultrymeal).Thebanmightprohibitfeedingofthematerialstoruminantsortoalllivestockspecies,ormightentirelyprohibituseofthematerial.

Insomecountries,afeedbanofruminantMBMtoruminantswasimplementedasthefirststep.ThebanwasthenoftenextendedtomammalianMBMduetothediffi-cultyindistinguishingbetweenheat-treatedMBMofruminantoriginandMBMofothermammalianorigin.Thisextendedbanwasgenerallyeasiertocontrolandenforce.

EvenwhennoMBMisvoluntarilyincludedincattlefeed,thereisstillariskofrecyclingtheagentthroughcrosscontaminationandcrossfeeding.ExperiencehasshownthatsmallamountsofMBMinfeedaresufficienttoinfectcattle.ThesetracesmayresultfromcrosscontaminationofMBM-freecattlefeedwithpigorpoultryfeedcontainingMBM,e.g.fromfeedmillsthatproducebothtypesoffeedinthesameproductionlines,fromtransportbythesamevehiclesorfrominappropriatefeedingpracticesonfarms.Apparently,usingflushingbatchesasasafeguardagainstsuchcrosscontaminationinfeedmills isnotsufficient.ThetracesofMBMincattlefeedthathavebeendetectedinEuropeancountriesaremostoftenbelow0.1%,whichseemstobeenoughtoinfectcattle.Therefore,aslongasfeedingofMBMtootherfarmedanimalsisallowed,crosscontaminationofcattlefeedwithMBMisverydifficulttoeliminate.Dedicatedproduc-tionlinesandtransportchannelsandcontroloftheuseandpossessionofMBMatfarmlevelarerequiredtocontrolcrosscontaminationfully. InmostEuropeancountries,abanonfeedingMBMtoallfarmanimalshasnowbeenimplemented.

MoredetailedinformationonmeasuresforlivestockfeedscanbefoundintheCapac-ityBuildingforSurveillanceandPreventionofBSEandOtherZoonoticDiseasesprojectcoursemanualentitledManagementoftransmissiblespongiformencephalopathiesinlivestockfeedsandfeeding(FAO,2007a).

rendering parametersRenderingofanimalby-products(e.g.bovinetissuesdiscardedattheslaughterhouse)andfallenstockintoMBM,whichisthenfedtoruminants,canrecycletheagentandallowamplification.Whenrenderingprocessesareproperlyapplied,thelevelofinfec-tivityisreduced.Ithasbeendeterminedthatbatch(ratherthencontinuous)renderingat133ºCand3barsofpressurefor20minuteseffectivelyreducesinfectivity(providingthat the particle size is less than 50 mm) although it does not completely inactivatetheagent(Tayloretal.,1994;TaylorandWoodgate,1997,2003;OIE,2005a).Therefore,using these parameters does not guarantee absolute freedom from infectivity in the

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MBM,especiallywhenmaterialwithhighlevelsofBSEinfectivityenterstherenderingprocess.

Moredetailed informationonmeasures forrenderingcanbe found in theCapacityBuilding forSurveillanceandPreventionofBSEandOtherZoonoticDiseasesprojectcoursemanualentitledManagementoftransmissiblespongiformencephalopathiesinlivestockfeedsandfeeding(FAO,2007a).

Specified risk materialsSpecifiedriskmaterials(SRM)aretissuesthathavebeenshown(orareassumed)tocontainBSEinfectivityininfectedanimals,andthatshouldberemovedfromthefoodand feedchains (TAFS,2004a). If thesematerialsareremovedatslaughterand thenincinerated, the risk of recycling the pathogen is markedly reduced. In addition, inordertoremoveinfectivityfurtherfromthefeedchain,carcassesfromhigh-riskcattle(e.g.fallenstock)shouldalsobetreatedasSRM.CountriesdefineSRMdifferently,anddefinitions sometimes change as new information becomes available, however mostdefinitionsincludethebrainandspinalcordofcattleover30months(Table1).

3.3. measures to prevent human exposureTheabovemeasurestoprotectanimalhealthindirectlyprotecthumanhealthbycon-trolling the amplification of the BSE agent. The most important direct measures forpreventinghumanexposure to theBSEagent in foodsaredescribed in the followingpages.

taBle 1. a summary of designated Srm in europe (as of october 200�)

Species and tissue european union uK and portugal Switzerland

age

Cattle

Skull(includingbrainandeyes) >12months - >6months

Entirehead(excludingtongue) - >6months >30months

Tonsils Allages Allages Allages

Spinalcord >12months >6months >6months

Vertebralcolumn(includingdorsalrootgangliabutNOTvertebraeoftailortransverseprocessesoflumbarandthoracicvertebrae) >24months >30months >30months(includestail)

Intestinesandmesentery Allages Allages >6months

Spleen - >6months -

Thymus - >6months -

Sheep and goatS

Skull(includingbrainandeyes) >12month >12months >12months

Spinalcord >12months >12months >12months

Tonsils >12months >12months Allages

Ileum Allages Allages Allages

Spleen Allages Allages Allages

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Ban of Srm and mechanically recovered meat for foodExcludingSRMandmechanically recoveredmeat (MRM) from thehuman foodchaineffectivelyminimizestheriskofhumanexposureandisthemostimportantmeasuretakentoprotectconsumers (TAFS,2004a).MRMisapastederived fromcompressedcarcasscomponentsfromwhichallnon-consumabletissueshavebeenremoved.Thesecarcasscomponentsincludebonesaswellasthevertebralcolumnwiththespinalcordanddorsalrootgangliaoftenattached.TheMRMisthenusedincookedmeatproducts,suchassausagesandmeatpies,and,ifruminantmaterialisincluded,isregardedasamajorBSEriskfactor.

BSe detection at slaughterMeasuresforminimizingrisksforhumanhealthrequiretheidentificationandelimina-tionofclinicallyaffectedanimalsbeforeslaughter,whichcanonlybeachievedthroughanadequatesurveillanceprogrammeincludinganantemorteminspectionspecificforBSE.BecausetheSRMfromclinicallyaffectedanimalsisknowntocontaininfectivity,removalanddestructionof theseanimalsprior toentering theslaughterhousehavetwoclearlypositiveeffects:

• Theriskofinfectivematerialenteringthefoodandfeedchainsisreduced.• There is lesscontaminationof theslaughterhouse,and lesspotential forcross

contaminationofnormalcarcasses.Inaddition,mostcountriesinEuropehavebeenconductinglaboratorytestingofall

slaughter cattle over 30 months of age (or even younger) for BSE since 2001 (TAFS,2004b).

Thebenefitsoftestingordinaryslaughtercattleare:• It identifies the very few positive animals that may not yet be showing clinical

signs.• Itdecreasestheriskofcontaminatedmaterialenteringthe foodchain in those

countrieswhereothermeasures(e.g.antemorteminspection,SRMremoval)maynotbeeffectivelyimplemented.

• Itcouldincreaseconsumerconfidenceinbeefandbeefproducts.• Itmayallowimportbanstobelifted(althoughsomeimportsbansmaybeinviola-

tionofWTOrules).

The drawbacks are:• Itisextremelyexpensive.• Itmaygiveafalsesenseofsecuritytoconsumers.• It may diminish the incentive to implement and enforce effectively other, more

effectivemeasures(suchasantemorteminspection).• Itcouldleadtoincreasedcontaminationwithinslaughterhousesduetoprocessing

ofagreaternumberofpositivecarcassesifothermeasuresarenotimplemented.AllcurrentlyavailablemethodsfordiagnosingBSErelyonthedetectionofaccumu-

latedPrPScinthebrainofinfectedanimals.Therefore,cattlemusthavealreadybeenslaughteredbeforeconfirmationofdiseasestatuscanbemade,potentiallyincreasingtheriskofcontaminationofcarcasseswithaninfectiousagent.Topreventthis,identi-ficationandremovalofclinicallyaffectedanimalsbythefarmerorveterinarianduringanantemorteminspectionareoptimalcontrolsteps.Laboratorydiagnostictestingiscoveredindepthinsubsequentchaptersinthismanual.

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measures to avoid cross contamination of meat with SrmIthasbeenshownthattheuseofcertaintypesofcaptiveboltgunstostuncattlepriortoslaughtercausesbraintissuetoenterthebloodstreamthatcouldbedisseminatedthroughout the carcass (including muscle). Therefore, pneumatic bolt stunning andpithingarenowforbiddenbymanycountriesinEuropeandelsewhere.Hygienicmeas-urestakenintheslaughterhousetoreducepotentialcontaminationofmeatwithSRMarealsoimportant.

MoredetailedinformationonSRMremovalandothermeatproductionissuescanbefoundintheCapacityBuildingforSurveillanceandPreventionofBSEandOtherZoonot-icDiseasesprojectcoursemanualentitledManagementof transmissiblespongiformencephalopathiesinmeatproduction(FAO,2007b).

3.4. on-farm measuresClassicalcontrolmeasuresforinfectiousdiseases(biosecurity,quarantine,vaccination)donotgenerallyapplytoBSE.Givenallavailableevidence,theBSEagentisnottrans-mittedhorizontallybetweencattlebutonlythroughfeed,primarilyingestionofcontami-natedMBMduringcalfhood.WhenaBSEcaseisdetected,ithasbeenshownthatothercattlewithinthatherdareunlikelytotestpositiveforBSE,despitethelikelihoodthatmanycalvesofsimilaragetothecaseallconsumedthesamecontaminatedfeed.

However,someon-farmstrategies,primarilythosethatfocusonfeedasasourceofinfection,andsomecullingprogrammesdocontributetothecontrolanderadicationofBSE.Cullingstrategiesvaryamongcountries,andoftenchangeovertime.Somediffer-entcullingstrategiesthathavebeenappliedinclude(SSC,2000;2002c):

• theindexcaseonly• allcattleonthefarmwheretheindexcasewasdiagnosed• allcattleonthefarmwheretheindexcasewasbornandraised• allcattleontheindexcasefarmandonthefarmwheretheindex casewasbornandraised• allsusceptibleanimalsontheindexcasefarm (includingsheep,goatsandcats)• “feed-cohort“(cattlethatcouldhavebeenexposedto thesamefeedastheindexcase)• “birth-cohort“(allcattlebornoneyearbeforeoroneyear aftertheindexcaseandraisedonthesamefarm)

Whileherdcullingmaybeapoliticallyexpedientmeansofincreasingconsumercon-fidenceandfacilitatingexports,itisunlikelytobeanefficientriskmanagementmeas-ure (Heim and Murray, 2004). There are significant problems in implementing suchastrategy.Farmerssee itasaradicalapproachbecause it results inaconsiderablewasteofuninfectedanimals.Althoughtheremaybesufficientcompensationforculledanimals, farmersmaynotbelieve it is reasonable tocullapparentlyhealthy,produc-tiveanimals.Inadditiontheyarelikelytolosevaluablegeneticlinesand/ortheir“life’swork”.Forthesereasons,farmersmaybelesswillingtonotifysuspectcasesifcullingoftheirentireherdcouldresult.

Evidencefromanumberofcountriesindicatesthat,inthoseherdswheremorethanonecaseofBSEhasbeendetected,theadditionalcase(s)werebornwithinoneyearof

Herdculling

Cohortculling

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theindexcase.Asaresult,cullingabirthcohortisamorerationalriskmanagementstrategyasitfocusesonthoseanimalswithinaherdthathavethegreatestchanceofhavingBSE.Evenso,dependingontheinitiallevelofexposureandtheoriginalsizeofthecohort,itislikelythatrelativelyfewadditionalcasesofBSEwillbedetectedinthebirthcohortofaherd indexcase.Cohortculling is,however, likely tobemuchmoreacceptabletofarmerswhencomparedwithherdculling.

3.�. import controlThebestmeansofpreventingtheintroductionofBSEistocontroltheimportofcertainBSEriskproductsfromcountrieswithBSEorcountriesthatareatriskofhavingBSE.Mostcountriesdonotbanimportsofpotentiallyinfectivematerialsuntiltheexportingcountry has reported their first BSE case. This is usually too late, however, becausethe risk already existed before the first case was detected. Materials that should beconsideredriskyforimport(unlessappropriatesafetyconditionsaremet)includeanymammalianderivedmeals (includingMBMandotherproteinmeals), feedcontainingMBM,livecattleandoffal. Importofbeefandbeefproductsforhumanconsumption,including processed beef products, whole cattle carcasses and bone-in beef, shouldalsobecontrolled,especiallyfortheexclusionofSRM.Debonedbeefmeatisgenerallyconsideredasnon-riskyforimport.

3.�. enforcementAlthough implementation of each measure decreases the overall risk of exposure,combining measures decreases the risk more profoundly (Heim and Kihm, 2003).Forexample,feedbansimplementedinconjunctionwithanSRMbanforfeedhaveastrongerimpact.Also,measuresmustbeeffectivelyimplementedandenforced.Simplyissuing a regulation or ordinance without providing the necessary infrastructure andcontrolswillnotachievethedesiredgoals.Educationofallpeopleinvolvedisrequiredatalllevelsandinallsectorsinordertoimproveunderstandingandcapacity,andthusimprovecompliance.

4. CliniCal SignSIncontrasttomanyBSEcasespicturedinthemedia,mostcattlewithBSEhavesubtlesignsofdisease.Signsareprogressive,variableintypeandseverity,andmayincludedepression,abnormalbehaviour,weightloss,sensitivitytostimuli(light,sound,touch)andgaitormovementabnormalities.Othersigns thathavebeennoted insomeBSEcasesincludereducedmilkyield,bradycardiaandreducedruminalcontractions(Braunetal.,1997).

DifferentialdiagnosesforBSEincludebacterialandviralencephalitides(e.g.bornadisease, listeriosis, sporadic bovine encephalitis, rabies), brain edema, tumors, cer-ebrocortical-necrosis(CCN),cerebellaratrophy,metabolicdiseasesandintoxications,aswellasothercausesofweightlossandneurologicalabnormalities.

Becausenoneof theclinical signsarespecific (pathognomonic) for thedisease,adefinitive clinical diagnosis cannot be made. With experience, however, farmers andveterinarianscanbecomeefficientatearlyidentificationofBSEsuspects.Thesesuspi-cionsshouldalwaysbeconfirmedthroughlaboratorytesting.

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�. SurveillanCe SyStemS�.1. objectives of surveillance ThetwomajorobjectivesforBSEsurveillancearetodeterminewhetherBSEispresentinthecountryand,ifpresent,tomonitortheextentandevolutionoftheoutbreakovertime. In this way, the effectiveness of control measures in place can be monitoredandevaluated.However, thereportednumberofBSEcases inacountrycanonlybeevaluatedwithinthecontextofthequalityofthenationalsurveillancesystemandthemeasurestaken.BSEriskcanstillexist inacountry,evenifnocasesarefoundwithsurveillance.Surveillanceaimstosupplementthemorecomprehensivedataprovidedbyariskassessment(HeimandMumford,2005).

GeneralguidelinesfordiseasesurveillanceandspecificguidelinesforanappropriatelevelofBSEsurveillanceforthedifferentcategoriesofnationalriskareprovidedintheWorldOrganisationforAnimalHealth(OIE)TerrestrialAnimalHealthCode(OIE2005b,c).TheserecommendationsareconsideredbyWTOandtheinternationalcommunityastheinternationalstandards(WTO,1994).

�.2. passive surveillanceInmostcountriesBSEislistedasanotifiabledisease,whichisabasicrequirementforafunctioningpassive(aswellasactive)surveillancesystem.However,somecountrieshavenonationalpassivesurveillancesystemforBSE,oronlyaweaksystem.

Until1999,BSEsurveillanceinallcountrieswaslimitedtothenotificationofclinicallysuspectedcasesbyfarmersandveterinarians(andothersinvolvedinhandlinganimals)totheveterinaryauthorities(passivesurveillance).Itwasassumedthatthiswouldallowearlydetectionofanoutbreak(HeimandWilesmith,2000).However,becausepassivesurveillancereliessolelyonthereportingofclinicalsuspectsandisdependentonmanyfactors,includingperceivedconsequencesonthefarmanddiagnosticcompetence,itisnotnecessarilyconsistentorreliable.Thus,althoughpassivesurveillanceisacrucialcomponent of any BSE surveillance system, it has become increasingly obvious thatpassivesurveillancealoneisnotsufficienttoestablishtherealBSEstatusofacoun-try.

Forapassivesystemtofunctioneffectively,severalfactorsmustbeinplace:veterinary structure:Thediseasemustbenotifiable.Case definition: A legal definition of BSE must exist and must be broad enough toincludemostpositivecases.disease awareness:Theappropriateindividuals(farmers,veterinarians)mustbeabletorecognizeclinicalsignsofthedisease.willingness to report:Theremustbeminimalnegativeconsequencestothe identifi-cationofapositivecaseatthefarmlevelandmeasuresmustbeconsidered“reason-able”.Compensation scheme: The costs of culled animals must be reasonably compen-sated.diagnostic capacity:Theremustbeadequatelaboratorycompetence.

Becausethesefactorsvarygreatly,bothamongcountriesandwithincountriesovertime,theresultsofpassiveBSEsurveillancesystemsaresubjectiveandevaluationandcomparisonofreportednumbersofBSEcasesmustbemadecarefully.

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�.3. active surveillanceTooptimizeidentificationofpositiveanimalsandimprovethesurveillancedata,thosepopulationsofcattlethatareatincreasedriskofhavingBSEshouldbeactivelytargetedwithinanationalsurveillancesystem.Withtheintroductionoftargetedsurveillanceofcattleriskpopulationsin2001,alargenumberofcountriesinEuropeandalsothefirstcountriesoutsideEuropedetectedtheirfirstBSEcases.

Cattlewithsignsofdiseasenon-specifictoBSEandcattlethatdiedorwerekilledforunknownreasonsmaybedefinedindifferentcountriesassickslaughter,emergencyslaughter,fallenstockordownercows.TheprobabilityofdetectingBSE-infectedcat-tleishigherinthesepopulations,asitmayhavebeenBSEthatledtothedebilitation,death, cull or slaughter of these animals. Many of these cattle may have exhibitedsomeoftheclinicalsignscompatiblewithBSE,whichwerenotrecognized.Theexpe-rienceofmanycountriesinthelastyearshasshownthat,afterclinicalsuspects,thisisthesecondmostappropriatepopulationtotargetinordertodetectBSE.Targetedsurveillanceaimstosamplecattleintheseriskgroupsselectively,andtestingoftheseriskpopulations isnowmandatory inmostcountrieswithBSEsurveillancesystemsinplace.

Healthycattle => Routineslaughter

Cattlewithnon-specificsigns(e.g.weightloss,lossofproduction)and => Sick/emergencyslaughter,cattlethatdiedforunknownreasons fallenstock,downercows(onthefarm,duringtransport)

CattlewithspecificsignsofBSE => BSEsuspects(orsuspicionofBSE)

Theageofthepopulationtestedisalsoimportant,astheepidemiologicaldatashowthatcattleyoungerthan30monthsrarelytestpositiveforBSE.Therefore,targetedsur-veillanceaimstosamplecattleover30monthsofageselectivelyintheriskpopulations,whichmaybeidentifiedonthefarm,attransportorattheslaughterhouse.

However,despite the fact that correctly implementedsamplingof riskpopulationswouldhypotheticallybesufficient toassessBSE inacountry, testingasubsampleofhealthyslaughteredcattleshouldbeconsidered.Thisisneededtominimizediversionofquestionablecarcassestoslaughter,i.e.toimprovecompliance.Iffarmersareawarethat randomsampling isoccurring,andwhen theprobabilityofbeing tested is largeenough,theyarelesslikelytosendsuspectanimalsdirectlytoslaughter.

Thespecificsurveillanceapproachesvaryamongthedifferentcountries.TheEUandSwitzerlandaretestingtheentireriskpopulationover24and30monthsofage,respec-tively.IntheEU,additionally,allcattlesubjecttonormalslaughterover30monthsofage are currently tested, whereas in Switzerland a random sample of approximately5%istested.CountriesoutsideEuropehaveimplementedavarietyofdifferenttestingsystems.FromtheexperiencesgainedinEurope,itisclearthatitismostefficienttoensuretheeffectiveimplementationofpassiveandtargetedsurveillanceinriskpopula-tionsratherthantofocusontestingoftheentirenormalslaughterpopulation.

Surveillance for TSEs is covered in depth in the Capacity Building for Surveillance

Riskgroups

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and Prevention of BSE and Other Zoonotic Diseases project course manual entitledEpidemiology,surveillanceandriskassessmentfortransmissiblespongiformencepha-lophathies(FAO,2007c).

�. riSK aSSeSSment�.1. BSe status and international standardsFora longtime,BSEwasconsideredaproblemexclusivelyof theUK.Evenafter thedetectionofBSEcasesinseveralcountriesoutsidetheUK,theriskofhavingBSEwascategoricallydeniedbymanyothercountries.Onlyaftertheintroductionofactivesur-veillancedidseveral“BSE-free”countriesdetectBSE.

Before2005,theOIEdescribedfiveBSEcategoriesforcountries,butinMay2005anewBSEchapterwasadopted(OIE,2005d)reducingthenumberofBSEstatuscatego-riestothefollowingthree:

• Country,zoneorcompartmentwithanegligibleBSErisk• Country,zoneorcompartmentwithacontrolledBSErisk• Country,zoneorcompartmentwithanundeterminedBSErisk

According to the OIE, a primary determinant for establishing BSE risk status of acountry,zoneorcompartmentistheoutcomeofascience-basednationalriskassess-ment.Thisassessmentmaybequalitativeorquantitative,andshouldbebasedontheprinciplesgivenintheCodeChapters1.3.1and1.3.2onRiskanalysisandtheAppendix3.8.5 on Risk analysis for BSE (OIE, 2005e,f,g). The OIE Code Chapter on BSE (OIE,2005d) lists the followingpotential factors forBSEoccurrenceandtheirhistoricper-spectivethatmustbeconsideredinsuchanassessment:Releaseassessment2

• theTSEsituationinthecountry• productionandimportofMBMorgreaves• importedliveanimals,animalfeedandfeedingredients• importedproductsofruminantoriginforhumanconsumptionandforinvivouse

incattleSurveillance for TSEs and other epidemiological investigations (especially surveil-

lanceforBSEconductedonthecattlepopulation)shouldalsobetakenintoaccount.Exposureassessment:

• recyclingandamplificationoftheBSEagent• the use of ruminant carcasses (including from fallen stock), by-products and

slaughterhousewaste,theparametersoftherenderingprocessesandthemeth-odsofanimalfeedmanufacture

• thefeedingbansandcontrolsofcrosscontaminationandtheirimplementation• thelevelofsurveillanceforBSEandtheresultsofthatsurveillance

In addition to an assessment of BSE risk, the OIE status categorization for BSEincludesevaluationofsomeofthemeasuresinplaceinthecountry.AccordingtotheOIECode,factorsevaluatedintheestablishmentofBSEstatusshouldinclude:

• theoutcomeofariskassessment(asdescribedabove)• disease awareness programmes to encourage reporting of all cattle showing

clinicalsignsconsistentwithBSE

2 In2006,theOIEBSEchapterwasmodifiedsothatonlyBSE,andnototherTSEs,isincludedintheexposureassessment.

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• compulsorynotificationandinvestigationofallcattleshowingclinicalsignscon-sistentwithBSE

• examinationinanapprovedlaboratoryofbrainsamplesfromthesurveillanceandmonitoringsystem

�.2. the geographical BSe risk assessmentThegeographicalBSEriskassessment(GBR)isaBSEriskassessmenttooldevelopedby the Scientific Steering Committee of the Europan Commission and based on OIEassessmentcriteria.TheGBRisaqualitativeindicatorofthelikelihoodofthepresenceofoneormorecattlebeinginfectedwithBSE,atagivenpointintimeinacountry,andhasbeenappliedtoanumberofcountriesthroughouttheworld.Themethodisaquali-tativeriskassessment,whichusesinformationonriskfactorsthatcontributeeithertothepotentialforintroductionofBSEintoacountryorregionortotheopportunityforrecyclingof theBSEagent inacountryorregion.The followingquestions,relatedtoreleaseandexposure,areansweredthroughtheGBR:

• Wastheagentintroducedintothecountrybyimportofpotentiallyinfectedcattleorfeed(MBM),andifsotowhatextent?

• Whatwouldhappeniftheagentwereintroducedintotheanimalproductionsys-tem,i.e.woulditbeamplifiedoreliminated?

Beforethedetectionofthefirstcasesinmany“BSE-free”countries,theGBRshowedthatariskcouldbepresent.Thisconfirmedtheconceptthataserious,comprehensiverisk assessment must be carried out to estimate the extent of the BSE problem incountries.

Thus, decisions on preventive measures should be based on such a detailed riskassessment, whether it is the GBR or another science-based assessment based onOIErecommendations.Nocountryshouldwaituntilthefirstcaseoccursbeforetakingpreventivemeasures.ThereremainmanycountrieswithanunknownBSErisk.Inordertominimizeimportrisksfromthesecountries,furtherriskassessmentsareneededtoevaluatetherealBSEdistributionworldwide.

RiskassessmentforTSEsiscoveredindepthintheCapacityBuildingforSurveillanceandPreventionofBSEandOtherZoonoticDiseasesprojectcoursemanualEpidemiol-ogy,surveillanceandriskassessmentfortransmissiblespongiformencephalophathies(FAO,2007c).

�. referenCeSBraun u, Kihm u, pusterla n, Schönmann m. 1997. Clinical examination of cattle with

suspectedbovinespongiformencephalopathy (BSE).SchweizArchTierheilk139,35-41

http://www.bse.unizh.ch/english/examination/htmlsklinischer.htm

defra(department for environment food and rural affairs, uK).2004.BSEpages.http://www.

defra.gov.uk/animalh/bse/index.html

department of health, uK. 2006. CJD-homepage. http://www.dh.gov.uk/PolicyAndGuidance/

HealthAndSocialCareTopics/CJD/fs/en

detwiler la, Baylis m.2003.Theepidemiologyofscrapie.RevscitechOffintEpiz22(1),121-143

eloit m, adjou K, Coulpier m, fontaine JJ, hamel r, lilin t, messiaen S, andreoletti o, Baron t,

Bencsik a, Biacabe ag, Beringue v, laude h, le dur a, vilotte Jl, Comoy e, deslys Jp, grassi

J, Simon S, lantier f, Sarradin p.2005.BSEagentsignaturesinagoat.Vet.Rec.156,523-524

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eC (european Commission).2004.Foodandfeedsafety:legislationconcerningTSE.http://europa.

eu.int/comm/food/food/biosafety/bse/legisl_en.htm

fao. 2007a. Management of transmissible spongiform encephalopathies in livestock feeds and

feeding.Coursemanual.ProjectCapacityBuildingforSurveillanceandPreventionofBSEandOtherZoonoticDiseases.Rome

fao. 2007b. Management of transmissible spongiform encephalopathies in meat production.

RomeCapacityBuildingforSurveillanceandPreventionofBSEandOtherZoonoticDiseases.

Rome

fao. 2007c. Epidemiology, surveillance and risk assessment for transmissible spongiformencephalophathies.Coursemanual.ProjectCapacityBuildingforSurveillanceandPreventionofBSEandOtherZoonoticDiseases.Rome

heim d, Kihm u. 1999. Bovine spongiform encephalopathy in Switzerland - the past and the

present.RevscitechOffintEpiz18(1),135-144

heim d, Kihm u. 2003. Risk management of transmissible spongiform encephalopathies in

Europe.RevscitechOffintEpiz22(1),179-199

heim d, mumford e.2005.ThefutureofBSEfromtheglobalperspective.MeatScience70,555-

562

heim d, murray n.2004.PossibilitiestomanagetheBSEepidemic:cohortcullingversusherdculling

–experiencesinSwitzerland.In:Prions:achallengeforscience,medicineandthepublichealthsystem,2nded.EdsHFRabaneau,JCinatl,HWDoerr.Karger,Basel,Switzerland.pp186-192

heim d, wilesmith J.w.2000.SurveillanceofBSE.ArchVirolSuppl.16,127-133

lasmezas Ci.2003.Thetransmissiblespongiformencephalopathies.RevscitechOffintEpiz22(1),

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oie (world organisation for animal health).2005a.Proceduresforthereductionofinfectivityof

transmissible spongiform encephalopathy agents. Terrestrial Animal Health Code, Appendix

3.6.3.http://www.oie.int/eng/normes/mcode/en_chapitre_3.6.3.htm

oie. 2005c, General guidelines for animal health surveillance. Terrestrial Animal Health Code,Appendix3.8.1.http://www.oie.int/eng/normes/MCode/en_chapitre_3.8.1.htm

oie.2005c.Surveillance forbovinespongiformencephalopathy.TerrestrialAnimalHealthCode,Appendix3.8.4.http://www.oie.int/eng/normes/MCode/en_chapitre_3.8.4.htm

oie. 2005d.Bovinespongiformencephalopathy.TerrestrialAnimalHealthCode,Chapter2.3.13.

http://www.oie.int/eng/normes/MCode/en_chapitre_2.3.13.htm

oie.2005e.Generalconsiderations(riskanalysis).TerrestrialAnimalHealthCode,Chapter1.3.1.


oie. 2005f. Import risk analysis. Terrestrial Animal Health Code, Chapter 1.3.2. http://www.oie.

int/eng/normes/MCode/en_chapitre_1.3.2.htm

oie. 2005g. Factors to consider in conducting the bovine spongiform encephalopathy risk

assessment recommended inchapter2.3.13.TerrestrialAnimalHealthCode,Appendix3.8.5.


prince mJ, Bailey Ja, Barrowman pr, Bishop KJ, Campbell gr, wood Jm.2003.Bovinespongiform

encephalopathy.RevscitechnOffintEpiz22(1),37-60

Schreuder Be, Somerville ra.2003.Bovinespongiformencephalopathy insheep?RevSci techOffintEpiz22(1),103-120

SSC (Scientific Steering Committee of the european Commission).2000.OpinionoftheScientific

SteeringCommitteeonBSE-relatedcullingincattle.Adoptedatthemeetingof14/15September

2000.http://europa.eu.int/comm/food/fs/sc/ssc/out138_en.pdf

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SSC.2001a.OpiniononhypothesesontheoriginandtransmissionofBSE.AdoptedbytheScientific

SteeringCommitteeatitsmeetingof29-30November2001.http://europa.eu.int/comm/food/fs/

sc/ssc/out236_en.pdf

SSC. 2001c. Opinion on the six BARB BSE cases in the UK since 1 August 1996. http://europa.

eu.int/comm/food/fs/sc/ssc/out237_en.pdf

SSC.2001b.SafetyofmilkwithregardtoTSE:stateofaffairs.http://europa.eu.int/comm/food/fs/

sc/ssc/out175_en.html

SSC.2002a.Thesafetyofbovineembryos.AmendmenttotheSSCopinionof18-19March1999

onthepossibleverticaltransmissionofbovinespongiformencephalopathy(BSE).http://europa.


SSC. 2002b. Update of the opinion on TSE infectivity distribution in ruminant tissues. Initially

adoptedbytheScientificSteeringCommitteeatitsmeetingof10-11January2002andamended

atitsmeetingof7-8November2002.http://europa.eu.int/comm/food/fs/sc/ssc/out296_en.pdf

SSC.2002c.OpinionoftheSSContheadditionalsafeguardprovidedbydifferentcullingschemes

under the current conditions in the UK and DE. Adopted on 11 January 2002. http://europa.


tafS(international forum for tSe and food Safety). 2004a.TAFSpositionpaperonspecifiedrisk

materials. http://www.tseandfoodsafety.org/position_papers/TAFS_POSITION_PAPER_SPECIFI

ED%20RISK%20MATERIALS.pdf

tafS. 2004b. TAFS position paper on testing of cattle for BSE – Purpose and effectiveness.

http://www.tseandfoodsafety.org/position_papers/TAFS_POSITION_PAPER_ON%20TESTING%2

0OF%20CATTLE.pdf

tafS. 2007. TAFS position paper on the safety of bovine milk and bovine milk products”.

http://www.tseandfoodsafety.org/position_papers/TAFS_POSITION_PAPER_ON_MILK.pdf

taylor dm, woodgate Sl.1997.Bovinespongiformencephalopathy:thecausalroleofruminant-

derivedproteinincattlediets.RevscitechOffintEpiz16,187–198

taylor dm, woodgate Sl.2003.Renderingpracticesandinactivationoftransmissiblespongiform

encephalopathyagents.RevscitechOffintEpiz22(1),297-310

taylor dm, fraser h, mcConnell i, Brown da, Brown Kl, lamza Ka, Smith gra. 1994.

Decontamination studies with the agents of bovine spongiform encephalopathy and scrapie.

ArchVirol139,313–326

wells ga.2003.PathogenesisofBSE.VetResCommun.Suppl1,25-28

wells ga, Scott aC, Johnson Ct, gunning rf, hancock rd, Jeffrey m, dawson m, Bradley r.1987.

Anovelprogressivespongiformencephalopathyincattle.VetRec121(18),419-420

wells gah, dawson m, hawkins SaC, green rB, dexter i, francis me, Simmons mm, austin ar,

horigan mw. 1994. Infectivity in the ileum of cattle challenged orally with bovine spongiform

encephalopathy.VetRec135(2),40-41

wells gah, dawson m, hawkins SaC, austin ar, green rB, dexter i, horigan mw, Simmons

mm. 1996. Preliminary observations on the pathogenesis of experimental bovine spongiform

encephalopathy. In: Bovine spongiform encephalopathy: the BSE dilemma. Ed. C.J. Gibbs,

SeronoSymposia,Norwell,USASpringer-Verlag,NewYork,Inc.pp.28-44

wells ga, hawkins Sa, green rB, austin ar, dexter i, Spencer yi, Chaplin mJ, Stack mJ, dawson

m. 1998. Preliminary observations on the pathogenesis of experimental bovine spongiform

encephalopathy(BSE):anupdate.VetRec142(5),103-106

wells ga, hawkins Sa, green rB, Spencer yi, dexter i, dawson m. 1999. Limited detection

of sternal bone marrow infectivity in the clinical phase of experimental bovine spongiform

encephalopathy(BSE).VetRec144(11),292-294

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wilesmith Jw, wells ga, Cranwell mp, ryan JB. 1988. Bovine spongiform encephalopathy:

epidemiologicalstudies.VetRec123(25),638-644

wilesmith Jw, ryan JB, atkinson mJ.1991.Bovinespongiformencephalopathy:epidemiological

studiesontheorigin.VetRec128(9),199-203

will rg, ironside Jw, Zeidler m, Cousens Sn, estibeiro K, alperovitch a, posers S, pocchiari m,

hofman a, Smith pg.1996.AnewvariantofCreutzfeldt-JakobdiseaseintheUK.Lancet347,

921-925

world trade organization. 1994.AgreementonSanitaryandPhytosanitaryMeasures.FinalActoftheUruguayRound,Article5.http://www.wto.org/english/docs_e/legal_e/15-sps.pdf

wrathall ae.1997.Risksoftransmittingscrapieandbovinespongiformencephalopathybysemen

andembryos.RevsciTechOffintEpiz16(1),240-264

wrathall ae, Brown Kf, Sayers ar, wells ga, Simmons mm, farrelly SS, Bellerby p, Squirrell

J, Spencer yi, wells m, Stack mJ, Bastiman B, pullar d, Scatcherd J, heasman l, parker J,

hannam da, helliwell dw, Chree a, fraser h. 2002. Studies of embryo transfer from cattle

clinicallyaffectedbybovinespongiformencephalopathy(BSE).VetRec150(12),365-37

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laboratory-based

surveillance and

epidemiology

laBoratory-BaSed SurveillanCe and epidemiology

Knowledgeonthetheorybehindmonitoringandsurveillanceforanimaldiseases,ondiagnostictestcharacteristicsandonsamplingapproachesisessentialfortheunder-standingandcorrectdesignofmonitoringandsurveillancesystems(MOSS),andfortheinterpretationofresults.

Thischapterofthecoursemanualaddressessomefundamentalconceptsrelatedtodisease surveillance, measures of disease frequency (prevalence, incidence), samplesizecalculationsfordiseasedetectionandprevalenceestimation,diagnostictestevalu-ation (sensitivity, specificity, predictive values) and measures of association (relativerisk, odds ratio). Additional information is available in the “Veterinary epidemiology–principlesandconcepts”and“SurveillanceforBSE”chaptersinthecoursemanualentitledEpidemiology,surveillanceandriskassessmentfortransmissiblespongiformencephalophathies(FAO,2007).

TheexercisesweredesignedforusewiththefreesoftwarepackageWinEpiscopev2;thissoftwarepackagecanbedownloadedfromvariousWebsites(forexample,EpiV-etNet,2006).

1. monitoring and SurveillanCe SyStemS The expression “Surveillance” goes back to the time of the French Revolution (lateeighteenthcentury)whenthistermdescribedanactivityofgovernmentalforces“…tokeepaneyeonsubversivesubjects…”,certainlywithanintentiontotakeactionwhendeemednecessary.

In more recent documents produced by the World Organisation for Animal Health(OIE)andotherinternationalbodies,acleardistinctionisbeingmadebetweenmonitor-ingandsurveillance:

monitoring (towatch, follow,observe):acontinuous (ongoing)processofdatacol-lection on the health status (health-related events) within animal populations over adefinedperiodoftime(couldpotentiallybe“forever”).

Surveillance(monitorandcontrol):extensionofmonitoringinwhichcontroloreradi-cationactionistakenonceapredefinedlevelofthehealth-relatedevent(“disease”)hasbeenreached.

Unfortunately,thisterminologyhasnotbeenusedconsistently;quitefrequentlythetermsurveillanceisusedverygloballytodescribeanyactivityrelatedtodetectingcasesof disease within populations. One of the reasons could be that (in veterinary publichealth)basicallyallanimaldiseasesthataremonitoredarealsoregulatedbycertaincontrol programmes. There, the use of surveillance is indeed appropriate. If, as anexample,theprevalenceofNewcastledisease(ND)inwildbirdsisroutinelyassessedbytestinghuntedandfounddeadbirdsbutnocontrolmeasuresareinplaceiftheagentisfound,thenthiswouldconstitutea“simple”monitoringapproach.

Reportingofclinicallysuspicious (sick)animalswas introduced firstregionallyandthennationallyduringthenineteenthcenturyinordertocontrolrinderpest.Themain

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reason was that veterinary authorities realized that individual animal owners did nothave the resources to prevent the spread of these diseases from heir livestock tootherfarms,thusresultinginlargeoutbreakswithhigheconomiclosses.Theconceptof mandatory reporting of clinical suspects and subsequent movement restrictions,destructionandcompensationforlosses(bytheauthorities),onceprovenforthatdis-ease,wasquicklyadoptedforotherinfectious(transmittable)animaldiseasessuchasanthrax, rabies, foot-and-mouth disease (FMD), contagious bovine pleuropneumonia(CBP),sheeppox,glanders,dourineandscabiesofsheepandhorses.Ithasremainedthecoreapproachinordertocontroloutbreaksofhighlycontagiousanimaldiseases.

Thelistofdiseasesnotifiabletotheinternationalauthorities(OIE,2005)areanexten-sionofthisearlierselectionofreportableandcontrollableanimaldiseases.LegislationoftheEuropeanUnionaswellascountry-specificlegislationmightincludeadditionaldiseasesnotlistedbytheOIEinordertoaccountforregionaldifferences.

1.1. Classifications of moSSDiseasemonitoringandsurveillancesystemscanbeclassifiedbasedondifferentcri-teria(DoherrandAudigé,2001):

• Reasonfordatacollection(objectives,whyaMOSSisimplemented)• Sourceofdataandtypeofinformation• Approachtodatacollection(passive,activeetc.)• Numberofdiseasesincluded(oneorseveral)• Geographicregion(local,national,international)• Target population (clinical suspect cases, infected animals, potentially exposed

animals,etc.)• Approachtoselection/sampling(wholepopulationordefinedsample)• Controlelement(autonomousorintegratedprogramme)

OneofthemostfrequentreasonsfortheimplementationofaMOSSisthedocumen-tationtoothersthatthediseaseofinterestisbelowacertainthresholdlevelinordertosupportthetradeofanimalsandanimalproducts.Otherreasonsincludetheneedtocontroladiseaseforitszoonoticpotential,fortheeconomiclossesthatitcauses,forriskanalysisandresearchpurposesorforitshistoricalimportance.Intimesoflimitedresources,veterinaryservicesshouldassesstheexistingMOSSprogrammesandmakeconsciousdecisionsas towhetherornotcertainprogrammesneed tobecontinued,andifadditionalprogrammesareneeded.

Thereisabroadrangeofactivitiesandinstitutionswhereinformationonthediseasestatusofindividualanimals,groupsofanimalsorthepopulationisgenerated(DoherrandAudigé, 2001).Often,however, information fromonlyoneor twodatasources isused todefine thediseasestatusofananimalpopulation.One reasoncouldbe thattheinformationiscollectedindifferentdatabasesthatareoperatedbydifferentinsti-tutions,and that theexchangeof informationbetween the institutions (and thereforedatabases)doesnotexist.Otherreasonsincludethelackofacommonanimalorfarmidentification (and tracing) system, making it impossible to link information reliablyfromdifferentdatabases,and thedifficulties incorrectlyweighingandpoolingMOSSdatafromdifferentsourcesintooneestimateontheprobabilityofaregionorcountrybeingdisease-free.

Oncecollectedandanalysed,thereisacertainspectrumof“customers”interestedintheseMOSSdata.This,toavaryingdegree,includestheirownandforeignveterinary

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services(trade,veterinarypublichealthinterventions),therespectiveindustries,univer-sities,themedia,thegeneralpublicandothers.OnehastobeverycarefulinthewayMOSSinformationiscommunicatedtothedifferentinterestgroupssincetheirlevelofunderstandingand thereforecorrectly interpreting theconveyed informationwill varysubstantially.

Themost fundamentalapproach tocollectinganimaldiseasedata fromrespectivetargetpopulationsiseitherthroughbaseline(passive)monitoringorthroughtargeted(active)samplingand testing.Sentinelnetworks formanotherapproach tocollectinghealth-relatedinformationfrompopulations;thiswillnotbeaddressedfurtherhere.

Baseline or passive “surveillance” is defined as the, often mandatory, reporting ofclinicalsuspectcasestotheveterinaryauthorities. Insomecountries, thelegislationdifferentiatesbetweenimmediatenotificationofadiseasesuspicionandroutinereport-ingofpastcases,oftenpermonth,quarteroryear–dependingonthedisease.Thissystemof“passive”reportingreliesontheawarenessoftheanimalownersandveteri-narypractitionersofthedisease,andtheirwillingnesstoreportasuspiciouscaseoncetheyrecognizeone.Thissystemhasalonghistory,wassuccessfulforacertainrangeofdiseases,usesaninfrastructure(farmers,veterinarians)thatisalreadyinplace(lowcost forthe individualdisease),andcancoverabroadrangeofdiseases.However, itcanonlybeusedfordiseasesthatpresentclearclinicalsigns,andworksbestforthosediseasesthatarehighlycontagiousandthusspread,andthathaveashortincubationperiod. Moreover, the approach often underestimates the true level of disease, andin some instances the disease can go undetected, or detected but not reported, forextendedperiodsoftime.Therefore,reportedcasesindicatethatthediseaseispresentatatleastthatlevel.Noreportingofcases,however,cannotautomaticallybetakenastheproofthatacountryorregionisindeedfreeofthedisease.Inordertounderstandbetterthesequenceofeventsthatneedstotakeplacebeforeaclinicallydiseasedani-malis“processed”withinsuchasystemandidentifiedasa“case”,onecanconstructaneventtreeandassignprobabilitiesofsuccesstoeachstepofthattree.AnexampleisgiveninDoherrandAudigé(2001).Fordiseaseswithun-specificclinicalsymptomsandsevereconsequences (for theowner), the reportinganddetectionprobabilitiesmightberatherlow.

Targeted (active) “surveillance” is defined as the ongoing (continuous) or periodic(onceorrepeated)scientificallybasedcollectionofsamples/dataonacertaindiseasefromapredefinedanimaltargetpopulation.Itisacost-intensiveapproachthatneedsagoodscientificallybaseddesign.Theresults,however,shouldberepresentativeforthetargetpopulation,i.e.acceptedasvalid.Thisapproachgenerallyworkswellifafastandinexpensivediagnostictestsystemisavailabletodetecttheconditionof interest,andifatargetpopulationcanreliablybeidentifiedinwhichtheeventofinterestislikelytobehigherwhencomparedtotheoverallpopulation.Ifnosuchtargetpopulationcanbeidentified,thenageneralpopulationsurveyneedstobeperformed,resultinginhighercosts.

1.2. prevalence and incidenceTheoutcomeofanyMOSScanbeexpressedasameasureofdiseasefrequency.Themostcommonmeasuresofdiseasefrequencyare:

• Prevalence• Incidencecount

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• Incidencerisk,cumulativeincidence• Incidencerate,incidencedensity

Prevalence is defined as the number of existing (measurable) events (cases) in adefinedpopulationatrisk(ofbeingacase)ataspecificpointintime(crosssection).InFigure1,thiswouldbethenumberofcases(thickredhorizontallines)dividedbythetotalnumberofanimals(allhorizontallines)atagivenpointintime(crosssectionsattimesA,B,C,…,I).ThelowestprevalenceismeasuredattimeD(0/16)whilethehighestprevalencevaluesaremeasuredattimesFandG(both6/22).

Incidenceingeneralrelatestothenumberofnewcasesobservedinapopulationatriskoveradefinedperiodoftime.

Incidencecountisjustthetotalnumberofcasesoverthattimeperiodnottakingintoaccount thenumberofanimalsat risk; inFigure1, the incidencecount for the timeperiodA-Iwouldbe6(thefirstcasewasnotnew–italreadyexistedatthebeginningofthetimeperiod).

Cumulativeincidence(risk),themostcommonlyexpressedincidence,isthenumberofnewcasesoveraspecifiedtimeperiod(numerator)dividedbythenumberofanimalsatriskofbecomingacaseduringthattimeperiod(denominator).Thenewcasesarecountedasfortheincidencecount(A-I:6).Thedifficultylieswithmeasuringthepopula-tionatrisk(denominator),especiallyinadynamicpopulationwithexitsandnewentries.Frequentapproachesareeithertotakethepopulationatriskpresentatthebeginningofthetimeinterval(A:16),thepopulationatthemidpointoftheinterval(E:22)ortheaveragepopulationduringtheinterval((A+I)/2:17.5).Theresultisexpressedasapro-portionforthespecifiedtimeperiod(month,year,etc.).

Newcasesfortheincidencedensity(rate)arecountedasbefore.Thedenominator,however,isnowanaccumulationofanimaltimeatrisk,andtheresultingincidencerate

FIGURE 1

Schematic representation of prevalence and incidence (see text for explanation)

A B C D E F G H I

CasesNo. animals

1 1 1 0 2 6 6 4 4 16 20 20 19 22 22 22 20 19

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expressesthenumberofnewcasesperanimaltime(months)atriskinthegivenpopu-lation.Thismeasureisrarelyusedinveterinarymedicinesinceexactdataonanimaltimeatriskarefrequentlynotavailable.

2. diagnoStiC teSt CharaCteriStiCSInanyMOSSapproach,thecharacteristicsofthediagnostictestorcombinationoftests,subsequentlyreferredtoasthediagnostictestsystem,isessentialtotheperformanceofthatMOSS.Thus,thedesigners,operatorsandrecipientsof informationshouldbeawareof thepropertiesand limitationsof thesystemusedto identityandtoconfirmdiseasedindividuals.

Intheirfieldmanualforveterinarians,CannonandRoe(1982)giveagooddescriptionofthemostimportantdiagnostictestcharacteristics(definition,calculationandinter-pretation); the equivalent chapter in the textbook by Thrusfield (1995) provides moretechnicaldetailsonthisissue.

Initially,diagnostictestdevelopersareprimarilyinterestedintheanalytictestprop-erties.Thesearedefinedastheanalyticsensitivity,i.e.thelowerdetectionlimitorthesmallest,stilldetectable,amountofthesubstancethatthetestissupposedtomeasure(antigen,antibody,chemical,protein,etc.),and theanalyticspecificity (crossreactionprofile),i.e.theabilityofthetestnottoreacttoorbindwithrathersimilar(instructure,etc.)othersubstances.

When applying tests to populations, however, we need to know their operationalproperties,mainlytheirabilitytoclassifycorrectlytrulydiseasedandtrulynon-diseasedindividuals.

2.1. diagnostic test sensitivity and specificityThediagnostictestsensitivity(SE)isdefinedastheproportionoftrulydiseased(“goldstandard”positive)individualsthatthetestcorrectlyclassifiesas(test)positive.Itcanalsobeexpressedasaconditionalprobabilityofatest-positiveoutcome(T+)giventhattheanimalisdiseased(D+):

SE=P(T+|D+)

Thediagnostictestspecificity(SP)isdefinedastheproportionoftrulynon-diseased(“goldstandard”negative)individualsthatthetestcorrectlyclassifiesas(test)negative.Itcanbeexpressedastheconditionalprobabilityofatest-negativeoutcome(T-)giventhattheanimalisnon-diseased(D-):

SP=P(T-|D-)

This information on the test results (pos/neg) in respective “gold standard” posi-tive and negative groups of animals (samples) is very often presented in 2x2 tables.Anexampleofsucha2x2tablefromWinEpiscopev2ispresentedinFigure2. Inthisexample,outof100trulydiseased(goldstandardpositive)individuals,90werecorrectlyclassifiedas(test)positive,resultinginasensitivityof90%(95%confidenceinterval84.1–95.9%).Of the100 trulynon-diseased (goldstandardnegative) individuals,95werecorrectlyclassifiedas(test)negative,resultinginaspecificityof95%(CI90.7–99.3%).

Averyimportant issueindiagnostictestevaluationisthedefinitionofwhatconsti-

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tutesthe“goldstandard(referencestatus)”classificationforinfectedandnon-infectedindividualsagainstwhichthenewtestisvalidated.Theabsolute(positive)goldstandardisthedemonstrationoftheinfectiousagentafter(known)naturalinfectionandclinicaldisease.Thiscouldbefromclinicallydiseasedanimals inanaturaldiseaseoutbreakfromwhichtheinfectiousagentwasisolatedbyculture.Alsopossibleasanabsolutegold standard is the demonstration of clear and unique pathological lesions. Otherindirectmeasuresofdisease(orexposure)suchasthepresenceofantibodiesinadif-ferenttestsystemaredefinedasrelativereference(goldstandard)tests.Experimentalinfections and the use of animals from historically known negative populations areconsideredasalternativepositiveandnegativegoldstandards,respectively.

OneexampleisthevalidationofthefirstthreerapidscreeningassaysdevelopedforBSE. The gold standard positive pool consisted of 300 brain samples of good qualityfromUKclinicalBSEcasesthatwereconfirmedbothbyhistologyand immunohisto-chemistry(IHC).Thegoldstandardnegativepoolconsistedof1000goodqualitybrainsamplesfromapopulationassumedtobehistoricallyfreeofBSEthattestednegativeboth in histology and IHC. These two groups clearly consist the extreme ends of thepossiblespectrum(withreferenceto levelsofdetectable“agent”),andwereselectedforgoodquality.Itthuswasoflittlesurprisethatthethreetestscorrectlyclassifiedallsampleswithinthistrial.However,inthegeneralpopulation,agentlevelsandsamplequalitywillshowmorevariation,therebyreducingtheoveralltestperformance.

IntheongoingevaluationofnewrapidtestsforBSE,testdevelopershavetodocu-mentonamuchlargernumberoffieldsamplesthatthenewtestsarecomparable(inperformance)withtheexistingvalidatedassays.ForfurtherdetailsseetherespectivereportsoftheEuropeanCommissionthatareavailablethroughitsWebsite.

FIGURE 2

example of a 2x2 table (from winepiscope v2; see text for explanation)

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2.2. format of test resultsInordertovalidatediagnostictestsusingthetraditional2x2tableapproach,testresultsneedtobedichotomous(0/1,neg/pos,no/yes).Forcertaintestssuchasagglutinationassays,immunohistochemistry,westernblot,virusisolationandstriptests,theresultisgeneratedinsuchaway,anddatacanbeuseddirectly.Fortestswithaordinal(dilutiontiter)orcontinuouslymeasuredoutcome(temperature,opticaldensity,chemino-lumi-nescence)suchasfromanIndirectfluorescentantibodytest(IFAT),ELISA,Redbloodcellcount (RBC)etc.,acutoffvalue isrequired toclassifya testresultaspositiveornegative.Onlyafterclassificationisatransferintoa2x2tablepossible.

TheselectionofthecutoffvalueforELISA,forexample,willinfluencewhetheranon-perfecttestwillgeneratemorefalsenegative(FN)results(highercutoff)ormorefalsepositive(FP)results(lowercutoff).Thiscanbedemonstratedbythehistogram(Figure3)of2000negative(greenbars)and200positive(redbars)samples.

Moving the cutoff value towards higher optical density (OD) values will reduce thenumber of false positive results and thus increase the test specificity. Reducing thecutoff value will result in fewer false negative test results and therefore higher testsensitivity.TheReceiver-Operating-Characteristic (ROC)curveapproachallowsvisualexploration of the possible sensitivity and specificity combinations over a range ofselectedcutoff(Greiner,1996a,b).

2.3. the diagnostic test users’ viewUsersofdiagnostictestshavedifferentquestionsthattheyshouldaskinrelationtotestperformance.

true prevalenceIfadiagnostictestwasusedtoassesstheproportionoftestreactorswithinasample,theresultwillbetheapparentortest-positiveprevalence(AP).Thequestionisnowwhat

FIGURE 3

frequency of eliSa optical density readings (see text for explanation)

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thetrueprevalence(TP)ofdiseaseinthepopulationis.Ifthetestindeedis100%sensi-tiveand100%specific,thenAPandTPwillbethesame.Ifthetestisnotperfect,theRogan-GladenestimatorusingtheAP,andknowledgeonthetestcharacteristics(SE,SP),canderivetheTP,andthevariancefunctioncanprovidethenecessaryinformationtocalculate95%confidenceintervals:

predictive valueIfweseeanindividualtestresult(suchasapositivepregnancyteststrip),wewillauto-maticallytrytoassesshowreliablethattestresultcanbe.Thisprobabilityofatesttogivethecorrectresultiscalledthepredictivevalueofanindividual(positiveornegative)test result. Predictive values are calculated separately for the test-positive and test-negativegroup.

Thepositivepredictivevalue(PV+) isdefinedastheproportionoftest-positive indi-vidualsthat istrulydiseased(“goldstandard”positive). Itcanalsobeexpressedasaconditionalprobabilityofhavingatrulydiseasedindividual(D+)giventhattheindividualistestpositive(T+):

PV+=P(D+|T+)

Thenegativepredictivevalue(PV-)isdefinedastheproportionoftest-negativeindi-vidualsthatistrulynon-diseased(“goldstandard”negative).Itcanalsobeexpressedasaconditionalprobabilityofhavingatrulynon-diseasedindividual(D-)giventhattheindividualistestnegative(T-):

PV-=P(D-|T-)

Predictivevaluesdependonthediagnostictestcharacteristics:• highSEfewerFNtestresultshigherPV-• highSPfewerFPtestresultshigherPV+

Predictivevalues,however,alsodependonthetrueprevalenceofthediseaseinthepopulationwherethe(tested)individualcamefrom:

• highprevalencehigheroverallprobabilitythatindividualisdiseasedhigherPV+

• lowprevalence higheroverallprobability that individual isnon-diseasedhigherPV-

TestcharacteristicsSEandSPareassumedtoberelativelystableacrossdifferentpopulationsandprevalenceranges,whilepredictivevaluesvarywiththepopulationandtheirspecificdiseaseprevalence.

Serial and parallel testingAcombinationoftestsisoftenusedinordertoclassifyindividualanimalscorrectly.Testscaneitherbeusedinseries(onlytest-positivereactorsfromthefirsttestareexaminedinasecondtest),orinparallel(allsamplesareexaminedintwoormoretests).

Asimpleexampleofaserial(sequential)combinationoftestsistheclinicalsuspect

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reporting (screening test)andsubsequent laboratoryconfirmationof thosesuspects.AnotherexampleistheuseofaBSErapidscreeningtestonslaughteredcattleandthesubmissionoftest-positivesamplestothereferencelaboratoryforconfirmation.

Inordertomaximizetheoverallperformanceofaserialtestcombination,the(first)screening test should have a very high sensitivity (>99%). This ensures that (almost)allpositiveindividualsarecapturedinthescreening,butitwillalsogenerateacertainproportionoffalsepositivetestresults.Thefollow-up(confirmatory)testneedsahighsensitivitybutaveryhigh(>99%)specificitytodistinguishclearlybetweentrulydiseasedandtrulynon-diseased(butscreeningtestfalsepositive)individuals.

Iftwoormoretestsareappliedtothesamesampleinparallel,thedecisionrulewillinfluencetheoveralltest(combination)performance:

• positiveifatleastoneofthetestsispositivefewerfalsenegativeshighersensitivityandlowerspecificity(morefalsepositives)

• positiveonlyifalltestsarepositivefewerfalsepositiveshigherspecificitybutlowersensitivity(morefalsenegatives)

ScenariosofexpectedresultsfromcombinationsinseriesandinparallelcaneasilybeexploredusingtheWinEpiscopemodule“Tests/Multiple tests”.Thismodule,how-ever,assumesthatthetestresultsofthetwoormoretestsareindependentfromeachother.Ifthe(underlyingbiological)reasonfortwotestsbeingwrongisthesame,thenthetestoutcomesarecorrelated,andusingmultipletestslosesitsefficiency(highercostswithoutmuchdiagnosticimprovement).

The topic of herd-level testing (and diagnostic test characteristics) is not furtherexplored in this brief introduction. For more information, other sources should beconsulted.

2.4. exercises2a. neospora studyA study of an ELISA to test for Neospora antibodies in cattle reported the followingresults:

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yes no

eliSa Pos 41 2

Neg 1 140

UsetheWinEpiscopemoduleTest/Evaluationtocalculatethefollowing:1. CalculatetheELISAsensitivityandspecificitywith95%confidenceintervals.2. Calculate thepredictive valueofapositive test result,aswellas thepredictive

valueofanegativetestresult.3. Wouldyouhavethesamepredictivevalueofapositiveandanegativetestifyou

usedthetestinapopulationwithonly5%oftrueprevalence(ofNeospora-relatedabortions)?

UsetheWinEpiscopemoduleTest/AdvancedEvaluationthecalculatethefollowing:4. Calculatepredictivevaluesusingapopulationof500cattle,theabovecalculated

estimates of sensitivity/specificity and a true prevalence of 5% (Module Test/AdvancedEvaluation).

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2B. testing for low prevalence diseases YouareworkingwithadiagnostictestfordiseaseXthathasaSEof99.5%andaSPof98%.Apply that test inapopulationof10000cattlewithaprevalenceof0.1% (10cases).

UseWinEpiscopeTest/AdvancedEvaluationtocalculatethefollowing:1. Howmanytruepositiveandhowmanyfalsepositivetestresultswouldyouexpect

fromthispopulation?Whatisthepositivepredictivevalueofthetest?2. How many true negative and how many false negative test results would you

expectfromthispopulation?Whatisthenegativepredictivevalueofthetest?3. Ifyouapplythesametesttoapopulationwithadiseaseprevalenceof0.01%(1

case),howmanytestpositivesampleswouldyouexpect?4. Is the total number test positives from (3) very different from the test positive

resultsin(1)?5. Canthistestbeusedtodifferentiatebetweenthetwoprevalencelevels?

2C. Combining tests YouuseascreeningtestwithaveryhighSE(99.8%)andamoderatespecificity(95%)andfollowuponallpositivetestresultswithahighlyspecific(99.9%)andrathersensi-tive(98%)confirmationtest(serialtesting).Test100000samplesfromapopulationwithaprevalenceofdiseaseof0.1%.

UsetheWinEpiscopemoduleTest/MultipleTeststocalculatethefollowing:1. Calculate the expected frequencies of results for test 1 and test 2 when used

independently,aswellaswhentheyareusedinparallelandinseries.2. How many initial reactive samples do you expect from test 1 in the serial

approach?Howmanypositivesamplesdoyougetafterapplicationofthesecond(confirmation)test?

3. Whatarethemaindifferencesbetweentheserialandtheparallelapproach?

3. Sampling iSSueSTheobjectiveofanimalhealthsurveys(aspartofaMOSS)istoassess,withanacceptedlevelofcertainty,whetherthediseaseispresentinagivenanimalpopulationand,ifyes,atwhichlevel.Themainquestionswecanaskare:

• “Detectionofdisease”–isthediseasepresentatagivenlevel?• “Maximumnumberpositives”–whatisthemaximumnumberofpositiveanimals

inthepopulationgiventhatarandomsampleofsizenwastestednegative?• “Prevalenceestimation”-whatisthelikelyprevalenceofdisease(withspecified

levelofprecision/error)inthetargetpopulation?Inordertoanswerquestions1and3onecouldsimplyexamineallindividualsinthe

targetpopulation.However,weareoftenlimitedbyresources,andwanttogetthesameanswerfromexamining“just”asample,i.e.asubsetofthetargetpopulation.Inordertodothat,thesubset(sample)needstobe“representative”ofthetargetpopulation.Intheorythiscanbeachievedbydrawingasimplerandomsamplefromthatpopulation.Twodescriptionsofthemeaningofrandomsamplingare:

• Thebestwaytodrawatrulyrepresentativesamplefromapopulationistohavethesubjectsincluded“bychance”.

• Asamplingprocedurethathasatrulyrandomcomponentprovideseachsubject

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withinthetargetpopulationwiththesameprobability (likelihood,chance) tobeincludedintothesample.

Therearevarioussamplingapproachesthattheoreticallyallfulfiltherequirementofbeingrepresentative–exceptforthefirstone:

• Conveniencesamplenotrandom,notrepresentative• Simplerandomsamplingtrulyrandomselectionofsubjects• Systematicsamplingrandomcomponent(startingpoint)• Stratifiedrandomsamplingrandomselectionwithinstrata(breed,age,etc.)• Clustersamplingrandomselectionofclusters(herds,regions)• Multistagesamplingcombination,oftenclusterandsimplerandomsampling

For further details on the advantages, disadvantages and requirements of thesesamplingtechniques,therespectivetextbooksandmanualsshouldbeconsulted.Thefurtherdiscussionof this topicwithin thiscoursemanual isrestrictedto trulyrepre-sentativesimplerandomsamplingaspresentedinthefieldmanualbyCannonandRoe(1982)andintherespectivemodulesinWinEpiscopev2.

3.1. detection of diseaseInordertoassesswhetheradiseaseispresentinagiventargetpopulation(of140000animals, for example) at a given (threshold) prevalence (0.1%) or not, a sample of acertainsizeneedstoberandomlyselectedfromthetargetpopulation.Thesamplesizecalculationwillensurethat–withaconfidenceof95%–thereisatleastoneinfectedanimalinthatsampleIFthetruepopulationprevalenceindeedis0.1%.Figure4showsthecalculationwithinWinEpiscoperesultsinarequiredsamplesizeof2963animals.

3.2. maximum number of positivesIf, as an example, you have examined a sample of 14 000 randomly selected (repre-sentative)animalsfromagiventargetpopulationof140000individuals,andall14000werenegative, themaximumnumberofdiseased(positive)animalsthat theoreticallycouldstillbepresentinthetargetpopulationcanbecalculated.

FIGURE 4

winepiscope results for sample size (see text for explanation)

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Using the respective module in WinEpiscope, it can be determined that with 95%confidencethemaximumnumberofpositiveanimalsinthepopulationof140000ani-mals,given that therandomsampleof14000animalswasnegative, is29 (or0.02%prevalence;Figure5).

FIGURE 5

winepiscope results for maximum number of positives (see text for explanation)

FIGURE 6

winepiscope results for prevalence (see text for explanation)

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3.3. prevalence estimationThediseaseprevalence–withasample–inagiventargetpopulationcanbeestimatedwithapredeterminedlevelofprecision(acceptederror).Weneedtoprovideourbestguessofthatprevalenceinordertodothesamplesizecalculation.

In this example with a population size of 10 000, an expected prevalence 1%, anaccepted (absolute) error of +/- 0.5% and a level of confidence of 95%, the requiredsamplesizeis1321(adjustedsamplesize)(Figure6).

Inallthesecalculationsanadditionalassumptionbesidesthesamplingpopulationbeing representative (random), is that the diagnostic test system used to detect thecaseswithinthesampleisperfect.Violationsofeitheroftheassumptionsresultintheneedforalargersamplesize!

3.4. exercises 3a. detection of diseaseYouwanttocalculatethesamplesizerequiredtodetectatleastonepositiveindividualinyoursample(with95%confidence)whentheprevalenceisassumedtobex%.

UsetheWinEpiscopemoduleSamples/DetectionofDiseasetocalculatethefollow-ing:

1. Calculate the required sample sizes for a population of 10 000 individuals andprevalence values of 20, 10, 1 and 0.1% (for the lowest prevalence, enter 10infectedanimalsinstead).

2. Calculatethesamplesizesforthesameprevalencevaluesasin(1),butuseapop-ulationsizeof100000 individuals(forthelowestprevalence,enter100 infectedanimalsinstead).

3. Compareanddiscusstheresultsof(1)and(2).

3B. prevalence estimationYouareaskedtospecifythenumberofgoldstandardpositivesamplesneededtoesti-matethediagnostictestsensitivityofanewtest.ThetestdeveloperassumesthatthetruetestSEwillbe99%.

UsetheWinEpiscopemoduleSamples/Estimatepercentagetocalculatethefollow-ing:

1. Howmanygoldstandardpositivesampleshavetobetested(outofanassumedlargepopulationof10000)inordertoestimatethatproportion(trueSE)withanmaximalacceptederrorof+/-1%(and95%confidence)?

2. Use the WinEpiscope Help Menu to look up the meaning of the adjusted (cor-rected)samplesize.

4. meaSureS of aSSoCiationEpidemiologyisdefinedasthestudyofthedistribution(occurrence)anddeterminants(risk factors) of health-related events (diseases) in populations. This section of thechapteraddressesoneofthecoreareasofepidemiology:themeasurementsusedtodescribetheassociationbetweendiseaseandpotentialriskfactors.

Oneofthenecessarystepsinidentifyingpotentialcauses(riskfactors)foradiseaseistoshowthatthisspecificriskfactorindeedhasastatisticallysignificant(numerical)association with the disease (outcome). Depending on the measurement scale, this

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could be either a statistically significant difference in the mean values of a continu-ouslymeasuredvariable(potentialriskfactor)betweentwoormoredistinctoutcomes(oftendiseasestatus),orasignificantlyhigherfrequency(proportion)ofonelevelofacategoricalvariable(potentialriskfactor)intheoutcomegroups.Thethirdalternativeisthesignificant(linear)correlationbetweenacontinuouslymeasuredriskfactorandacontinuouslymeasuredoutcomevariable.

Inthecontextofthiscoursemanualthediscussionislimitedtothatofacategorical(binary)riskfactorandacategorical(binary)outcome,i.e.diseasestatus.

4.1. the basics of hypothesis testingIn statistical hypothesis testing, one assumes that in the overall population a nullhypothesis(H0)ofnodifferenceinthefrequencyofanevent(riskfactor)betweentwogroups(diseasedandhealthy)exists.ThealternativehypothesisHA,orstudyhypoth-esis,thatistestedagainstthenullhypothesis–eitheronesided(a>b;a<b)ortwosided (a<>b)–states that there isasignificantdifferencebetween the twooutcomecategories.Theteststatisticcalculatestheexpectedrangeofoutcomevaluesassum-ingthatH0istrue,andcomparesthesevaluesandtheirrelatedprobabilitieswiththeobservedstudyvalues.IfthestudyvaluesareveryextremeincomparisontothevaluesexpectedunderH0,i.e.theprobabilityofobservingexactlythestudyvalues–givenH0istrue–is<5%orp<0.05,thenoneconcludesthatH0canberejectedinfavourofHA.Thisvalueofp<0.05isthegenerally-acceptedlevelofstatisticalsignificance.

4.2. errors and p-valueThevalueofαdefinestheprobabilityofdecidingthatthereisasignificantassociationbetween potential risk factor and disease while there truly is no association (Type Ierror).Thep-valueof0.05or5%definestheproportionoftimesthatmakingsuchanerrorisacceptable.Similarly,aTypeIIerrorisdefinedastheprobabilityofmissingasignificantassociationinthestudywhenonetrulyexists.Thepowerofastudy istheprobabilityofastudy(design)tofindsuchanexistingassociation(Figure7).

FIGURE 7

types of errors

TrueStatus

Studydecision

H0

HA

H0

1 - α

αType I error

HA

βType II error

1 - βPower

33

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4.3. example Thefollowingdiscussionisrestrictedtothesituationofacategorical(binary)riskfac-torandacategorical(binary)outcomevariable–theeasiestcase.InThrusfield(1995),Table14.6(page211)displaystheresultsofastudyofurinaryincontinenceinasampleofspayed(castrated)andentire(non-spayed)caninefemales.

Spayed

Entire

Total

Incont.(disease)

34(a)

7(c)

41(a+c)

Normal

757(b)

2427(d)

3184(b+d)

Total

791(a+b)

2434(c+d)

3225(n)

Thestandardstatisticaltestusedtoassesswhetherthereisanassociationbetweentherow (potential risk factororexposurevariable)and thecolumn (disease)variableis the Chi-Square Test (χ2 test). It compares the observed frequencies in a 2x2 tablewith the frequenciesexpectedunder thenullhypothesis (that there isnoassociationbetweentherowandcolumnvariables).Therespectiveformulais:

Thedegreesoffreedom(df)fortheteststatisticare(rows–1)*(columns–1),i.e.inthisexampledf=(2-1)*(2-1)=1.Theassociatedcutoffχ2valueforstatisticalsignificanceis3.84,whiletheteststatisticresultfortheurinaryincontinenceexampleis76.01

Based on this value, it can be concluded that there is a significant associationbetweenspayingandurinaryincontinence.However,thisteststatisticgivesusneitheradirectionoftheassociation(positivevsnegative)noragoodestimationofitsstrengthofit–itjustsaysthatthereisone.

4.4. relative risk and odds ratioTwootherepidemiologicalmeasuresofassociation,therelativerisk(RR)andtheoddsratio(OR),providemoreinformationbothonthedirectionandstrengthoftheassocia-tion.Eachmeasurehasapossiblerangebetweenzeroandinfinity.Ifboththe(riskfac-tor)exposedandnon-exposedindividualshavethesameriskoroddsofdisease,thentheratiobetweenthetworisksoroddsis1,indicatingnoassociation(noinfluenceoftheriskfactoronthedisease).

The RR (depending on the situation also called prevalence ratio, rate ratio or riskratio)isdefinedastheRisk(D|E)/Risk(D|NE)andcanbecalculatedbythefunction:

RR=[a/(a+b)]/[c/(c+d)]

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Intheurinaryincontinenceexample,theRR=14.95(95%CI6.65–33.58)(calculatedinWinEpiscopev2).Inwords,thiswouldmeanthattheindividualriskofurinaryincon-tinenceinthisstudysamplewas14.95timeshigherforspayeddogswhencomparedtonon-spayeddogs.The95%confidenceinterval(extrapolationtolargepopulation)is6.65–33.58.

TheOR(alsocalledrelativeoddsorcross-productratio)isdefinedastheOdds(E|D)/Odds(E|ND)ortheOdds(D|E)/Odds(D|NE).Itcanbecalculatedbythefunction:

OR=[a/c]/[b/d]=[a/b]/[c/d]=a*d/b*c

Intheurinaryincontinenceexample,theOR=15.57(95%CI6.88–35.27)(calculatedinWinEpiscopev2).Inwordsthiswouldmeanthattheindividualodds(chance)ofuri-nary incontinence inthisstudysamplewas14.95timeshigherforspayeddogswhencompared to non-spayed dogs. The 95% confidence interval (after extrapolation to alargepopulation)is6.88–35.27.

TheRRandORbecomerelativelysimilarintheirabsolutevalueswhentheprevalenceof the outcome (disease) is <5%. They will always point in the same direction. Bothmeasures are not statistically significant when the 95% confidence interval includesthevalueof1,andtheRRisnotavalidmeasureforcase-controlstudies.Forfurtherdetails on study design and the appropriate measures please consult the respectiveepidemiologicaltextbooksshouldbeconsulted.

4.�. exercises 4a. association between neonatal deaths or culls in calves and the serum gamma globulin levelThegammaglobulin levelwasmeasured inperipheralbloodsamples thatwerecol-lectedwithinthefirst24hoursafterbirth.Theoutcome(whethertheysurvivedthefirstsevendayspostpartumornot)wassubsequentlyrecorded.

Theresultsarecross-classifiedinthefollowingtable:

gglob < 6.2%

gglob > 6.2%

Total

Died orculled

12

6

18

Survived

61

214

275

Total

73

220

293

UsetheWinEpiscopemoduleAnalysis/Cross-sectionaltocalculatethefollowing:1. Derive the appropriate measures of association (RR/OR) with 95% confidence

intervalsofneonataldeathforthe“exposed”group(gglob<6.2%)whencomparedtothe“non-exposed”group(gglob>6.2%).ATTENTION:Thetableset-upinWinE-piscopeisdifferentfromthewaythedataarepresentedabovesothecolumnsandrowsneedtobereversed.

Interprettheseresultsinwords.

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2. Whatisthevalueof“noassociation”forboththeRRandtheOR?Askedinanotherway:WhatwouldbetheRRorORthatweexpectifthereisnodifferenceintherisk(orodds)betweenthegroups?

3. Wedonotgetap-valuefromWinEpiscopeforthecalculatedRRandOR.Whichother informationprovidedbythepackagetellsuswhethertheobservedRRorORarestatisticallysignificant?

�. referenCeS Cannon rm, roe rt. 1982. Livestock disease surveys: a field manual for veterinarians. pp. 1-

35. Department of Primary Industries & Bureau of Rural Science, Australian Government

PublishingService,Canberra

doherr mg, audigé l.2001.Monitoringandsurveillanceforrarehealth-relatedevents-areview

fromtheveterinaryperspective.PhilTransRSocLondB356,1097-1106

epivetnet. 2006.Epidemiologicalanalysis.http://www.vetschools.co.uk/EpiVetNet/Epidemiological_

analysis_software.htm

fao. 2007. Epidemiology, surveillance and risk assessment for transmissible spongiformencephalophathies.Coursemanual,Project.CapacityBuildingforSurveillanceandPreventionofBSEandOtherZoonoticDiseases.Rome

greiner m. 1996a. Two-graph receiver operating characteristic (TG-ROC): a Microsoft-EXCEL

template for theselectionofcut-offvalues indiagnostic tests.J ImmunolMethods185,145-

146

greiner m.1996b.Two-graphreceiveroperatingcharacteristic(TG-ROC):updateversionsupports

optimisationofcut-offvaluesthatminimiseoverallmisclassificationcosts.JImmunolMethods191,93-94

greiner m, gardner ia.2000.Applicationofdiagnostictests inveterinaryepidemiologicstudies.

PrevVetMed45[1-2],43-59

oie (world organisation for animal health).2005.DiseasesnotifiabletotheOIE.http://www.oie.

int/eng/maladies/en_classification.htm

thrusfield m.1995.Veterinaryepidemiology.SecondEdition.BlackwellPublishing,Oxford,UK

�. SuggeSted Software (Can Be downloaded from www.vetSChoolS.Co.uK/epivetnet/)WinEpiscopev.2http://www.vetschools.co.uk/EpiVetNet/Sampling_software.htmSurvey Toolbox (with manual) http://www.vetschools.co.uk/EpiVetNet/Sampling_soft-ware.htm

�. SolutionS for eXerCiSeS 2a.1:SE=97.6%(93–100%);SP=98.6%(96.7–100%)2a.2:PV+=95.3%(89.1–100%);PV-=99.3%(97.9–100%)2a.3:No,PV+andPV-dependontestcharacteristicsandonprevalence.InapopulationwithalowerprevalenceonewouldhavealowerPV+andahigherPV-.2a.4:PV+=78.6%;PV-=99.9%2B.1:TP=10;FP=200;PV+=2.1%2B.2:TN=9,790,FN=0;PV1=100%2B.3:Testpositives=201-comparedto210inB.1.

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2B.4:Differenceis9test-positiveanimals,rathersmall.2B.�:No,thetestSEandSParestillnothighenoughtodifferentiatetheprevalences.2C.1:Lookattheoutcomeofthedifferentcalculations,compareSE’s,SP’sandPV’s!2C.2: 5095initialpositives(test1),200confirmedpositives(combinedtests)2C.3: Serial approach: only test 1 positives are tested in test 2, while in theparallel approach all samples are tested in both tests. Overall SE is higher inthe parallel approach while overall SP is higher in the serial approach. This,however, will always depend on the test characteristics of the selected tests.

3a.1:N=10000 prevalence=20%/n=14,10%/29,1%/294,0.1%/25883a.2:N=100000 prevalence=20%/n=14,10%/29,1%/298,0.1%/29503a.3:Samplesizesherearealmostindependentofpopulationsize,butincreasewithdecreasingprevalencevalues.3B.1:IfthetrueSEis99%thena(adjusted)samplesize=381goldstandardpositiveanimalswillbeneededtoestimatethatSEwithanerrorof+/-1%.3B.2: WinEpiscope HELP/INDEX/2.3 -> look for sampling fraction/corrected samplesize.

4a.1:OR=7.02(2.5–19.5);RR(PrevalenceRatio)=6.03(2.4–15.5)Inwords, thiswouldmeanthat the individualodds (chance)ofneonataldeath in thisstudysamplewas7.02timeshigherforcalveswithlowggloblevelswhencomparedtocalveswithhighlevels.The95%confidenceinterval(extrapolationtolargepopulation)is2.5–19.5.Similarly, the individualrisk (probability)ofneonataldeath in thisstudysample was 6.03 times higher for calves with low gglob levels when compared withcalveswithhighlevels.The95%confidenceinterval(extrapolationtolargepopulation)is2.4–15.5.4a.2:Thevalueof1(unity).4a.3:Ifthecalculated95%confidenceintervaldoesNOTincludethevalueofnoasso-ciation(i.e.1)thenweconsiderthemeasureofassociationasstatisticallysignificant.ThisdoesNOTautomaticallymeanthatthisassociation isalsobiologically important– thatneedstobeassessedthroughbiological thinkingandother indicationsof true(disease)causality!

3�

BiosafetyBioSafety

1. general ConCeptSMicroorganisms can be classified according to their pathogenicity for humans andanimals. According to this classification, precautions must be taken when handlingthem.Theseprecautionsarenecessaryprimarilytoprotectthepeoplehandlingtheseagents,butalsotoprotectthegeneralhumanpopulationandlivestockfromaccidentalexposure.Severalguidelineshavebeenpublishedontheclassificationsystemforandthehandlingofmicroorganisms.AninternationallywellacceptedguidelineistheWHOLaboratorybiosafetymanual (WHO,2003).Thismanualdefinesriskgroups,biosafetylevels(BL)oflaboratories,therequirementsforriskassessmentsandtherequirementsforeachofthebiosafetylevels.

WHOdefinesfourriskgroups,classified1to4,whicharebasedontheriskaspecificmicroorganismposesforhumansoranimals.Riskgroupsare:

1) Posesnooralowriskforindividuals(laboratorypersonnel)andthecommunity.2) Posesamoderateindividualriskandalowcommunityrisk.Effectivetreatmentor

preventionisavailable,andspreadofthemicroorganismisordinarilylimited.3) Poses a high individual risk and a low community risk. Effective prevention or

treatmentisavailable,butspreadofthemicroorganismisnotordinarilylimited.4) Posesahighindividualandahighcommunityrisk.Preventionandtreatmentare

notavailable,andthemicroorganismisreadilytransmitted.Theclassificationofmicroorganismsisbasedonfourcriteria:pathogenicity,trans-

mission,preventivemeasuresandtreatmentasfollows:pathogenicity is the ability of an organism to invade a host and to cause disease.

Microorganismswithahighpathogenicityaremore likely tobeclassified inahigherriskgroupandmicroorganismswithalowpathogenicityaremorelikelytobeclassifiedinalowerriskgroup.

transmission of microorganisms can, for example, be through direct contact, bewater-borne or be air-borne (by aerosol). When transmission of a microorganism islimitedtodirectcontact,thepossibilityofbecominginfectedisrelativelylow.Air-bornemicroorganismshaveahigherpossibilityofinfectinghosts.Also,thechanceofbecom-inginfectedishigherforlaboratorypersonnelwhoworkinclosecontactwithmicroor-ganismsthanforthecommunity.Themoreeasilymicroorganismscanbetransmittedthemorelikelytheyareclassifiedinahigherriskgroup.

preventive measures against microorganisms include vaccination. There can be adifferenceintheneedforpreventivemeasuresbetweenlaboratorypersonnelandthecommunity. There are no vaccinations available against some microorganisms, andthesemicroorganismsaregenerallyclassifiedinahigherriskgroup.

treatment.Ifnotreatmentisavailableforinfectionwithamicroorganism,thatmicro-organismisclassifiedinahigherriskgroup.

AlthoughWHOdefinestheriskgroupsandthecriteria, itdoesnotclassify thedif-ferent microorganisms into risk categories. It does give the requirements for a riskassessment,basedonwhichmicroorganismscanthenbeclassified.Singlecountries

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orregions(e.g.theEU)shouldperformsuchriskassessmentsandestablishanationalorregionalriskgroupclassificationforeachmicroorganismofinterest.

Dependingontheclassificationofthemicroorganism,precautionsshouldbetakentoprotectlaboratoryworkersandthecommunityfrompossibleinfection,forwhichWHOhasdefinedfourbiosafetylevelcategories.Thesecategoriescorrelatesomewhatwiththeriskgroupcategories,butalsoreflectwhatisbeingdonewiththemicroorganisminthelaboratory.

Bl 1: This level is the basic safety level for laboratories. Agents (microorganisms)handledatthisleveldonotordinarilycausehumandisease.Bothresearchandteachingcanbedoneinthislevellaboratory.

Bl 2:Thislevel isthesecondbasicsafetylevelforlaboratories.Agentshandledatthislevelcancausediseaseinhumans,buttheirpotentialfortransmissionislimited.Bothdiagnosticsandresearchcanbedoneatthislevel.

Bl 3:Thislevelrequiresacontainmentlaboratory.Agentshandledatthislevelcanbetransmittedbyaerosolandcancauseseriousinfections.Atthislevelspecialdiag-nosticsandresearchcanbedone.

Bl 4:Thislevelrequiresmaximumcontainment,andmaybeconsideredalaboratoryfordangerouspathogens.Agentsthatarehandledatthislevelmayposeahighriskoflife-threateningdisease,maybetransmittedasanaerosol,andmayhavenoassociatedvaccineortherapyavailable.Theseagentsareoftenconsideredexotictoacountry.

2. BioSafety for tSesIn2000, theEUpublishedadirectiveregulating theprotectionofworkers fromrisksrelated to work-related exposure to biological agents (EU, 2000), based on the WHOguidelines. In this directive, a new risk group is defined for BSE and related animalTSEs.CharacteristicsoftheBSEagentincludealimitedriskforlaboratorypersonnelandthecommunity;howeveraerosoltransmissioncannotbeexcluded(thoughithasneverbeendescribed).Thisnewriskgroupiscalled3**,whichmeansriskgroup3withsomealleviations.Scrapie,ontheotherhand,isstillclassifiedasriskgroup2.Accord-ingtotheSwissExpertCommitteeforBiosafety(2006),differentBLsarerequiredwhenhandlingBSEmaterials,dependingonthetypeofmaterial.Forexample,histologyandIHC on formic acid inactivated BSE material can be performed in a BL 1 laboratory,and for routine BSE diagnostics the laboratory should be BL 2 with some additionalmeasures.Areference laboratory forTSEmustbeBL3,butsomemodificationsareallowed.Attentionshouldbepaid to the fact thatBSE laboratory requirementsoftendifferbetweencountries.Ingeneral,thefollowingcriteriashouldbeconsidered:

• All BSE laboratories should have a separated working area with documentedrestrictedaccessthroughdoubledoors.Theanteroomcreatedbythetwodoorscanalsobeusedasachangingroom.Theoutsidedoorshouldbelabelledwitha“biohazard”sign.

• Astandardvectorcontrolprogramme(e.g.forants,flies)shouldbeinplaceforthelaboratory.

• Allsurfaceswithinthelaboratorymustberesistanttoacids,basesanddisinfect-antsbecauseallworkspacesshouldregularlybedecontaminated,whichcanonlybeaccomplishedusingstronglyoxidizingsubstances.

• The laboratory should have a class 2 biosafety cabinet (BSC) with a verticalupwardairflowsystemandafilterthatfiltersoutthesmallestpossibleparticles.

3�

BiosafetyThisairflowsystemensuresthataerosols,possiblycontainingBSE-infectedpar-ticles,areremovedtoreducethechanceofexposureoflaboratorypersonnel.Tomaintaintheupwardairflow,minimalequipmentshouldbeplacedintheBSC.

• Topreventcrosscontamination,acompletesetofdevicesandinstrumentsshouldbeavailable,solelyusedforBSEdiagnostics.

• Anautoclavethatcanachieveatemperatureof134°Cat3barofpressureshouldbepresent,optimally in the laboratory,butatminimumwithin thesamebuild-ing.

• LaboratorypersonnelshouldputonprotectiveclothingpriortoenteringtheBSElaboratory.Thisclothingcanbeputoverthenormalclothing.Standardprotectionincludes disposable overalls or gowns, protective gloves, protective glasses orfaceshields,anddedicatedlaboratoryshoesorshoecovers.Thisclothingshouldnotleavethelaboratoryexceptforfinaldisposal,andthenonlyafterautoclavingfordecontamination.

• When working in the BSC, personnel should wear a second pair of gloves andprotectivesleevestopreventanycontactbetweennormalclothingandBSEmate-rials.ThissecondpairofglovesshouldbeusedonlywhileworkingintheBSCandshouldbedisposedofdirectlyafterwards.

• Toensureastandardlevelofqualityandsafety,theBSElaboratoryshouldfollowgoodmicrobiologicaltechnique(GMT);(WHO,2003)guidelinesandimplementaqualityassuranceprogramme.

• Eating,drinkingandsmokingshouldnotbeallowedinthelaboratories.• Whathasbeendonewhen,bywhomandbasedonwhichinstructionsshouldbe

documentedfullyinwriting,allowingback-tracingwhennecessaryandfacilitat-ingaccuratejobassignments.

• Disposablematerialshavetheadvantagethattheydonothavetobedecontami-natedandre-used,andthereforereducethechancesforcrosscontamination.

• Thenumberofsharpobjectsusedshouldbeminimizedtoreducebreakageandpossibleinjuries.

• Equipmentontheworksurfacesshouldbeminimizedtoprovideadequatespaceforplacingsamplesanddecreasetheriskofaccidents.

• TheBSCshouldbeusedtopreventaerosolsintheworkingarea,evenwhenopen-ingatesttubeorduringcentrifuging.

• Pipettingshouldalwaysbedoneusingapipettingballoonoranautomatedsys-tem,neverbymouth.

• AllmaterialsthathavebeenincontactwithBSEsamplesshouldbeconsideredcontaminated.This implies thateverything that leaves the laboratoryshouldbedecontaminatedbeforedisposal.Decontaminationisaveryimportantstepwhenworking with BSE infected materials. Materials that have been in contact withinfectious material must either be disposed of directly in the trashcan (whichis decontaminated before disposal) or must be decontaminated when leavingtheBSC.Materialscanbegroupedinfourgroupsandmustbedecontaminatedaccordingly.1. Solidwaste.Solidwasteshouldbecollected inclosed trashcans,whichcan

beopenedbyfoottopreventhavingtotouchthetrashcanwhendisposingofmaterials.Withinthetrashcantheremustbeanautoclavabletrashbag,whichhas to be labelled “biohazard”. The trashcan should be emptied at regular

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intervals, and transport to the autoclave must be validated by documentingwho disposes of what in which way and along which route. Critical controlpoints along this route can be identified, analysed and adapted accordingly.Solidwasteshouldeitherbeautoclavedat134°C,at3bar foronehour (orunderotherinternationallyacceptedcircumstances)orincinerated.

2. Liquidwaste.Liquidwastemustbeincineratedorautoclavedunderthesameconditionsassolidwastewheneverpossible.However,practically,notallliquidwastecanbeincineratedorautoclaved.Inthiscase,liquidwastecanbeincu-batedwith2NNaOHforonehour.Itisimportantthatthefinalconcentrationis2N,thereforetheamountofNaOHaddedshouldbeadaptedtotheconcentra-tionoftheliquidwaste.

3. Instruments. Whenever possible instruments should be autoclaved accord-ingto theprocedurementionedabove. If this isnotpossible, theyshouldbeimmersedforonehourin4%NaOClorin2NNaOH.Bothmediaarestronglyoxidizing, however, so depending on the instruments this decontaminationprocedurecanbedetrimentaltothem.

4. Equipment and surfaces. The only way to decontaminate large equipmentand surfaces is to bring them in contact with paper towels soaked with 4%NaOClor2NNaOHforonehour.Afterwards,theyshouldberinsedwithwaterfor neutralization. The laboratory itself (floor, walls, shelves, etc.) should bedecontaminated at regular intervals and the BSC working area should bedecontaminatedaftereachuseusing4%NaOClor2NNaOH.

Althoughextensiveinformationonbiosafetyhasbeenpresentedinthischapter,itisclearthatsituationsnotdescribedheremaystillarise.UsingthisbackgroundonhowtohandleBSEinfectedmaterialsandequipmentwilllikelyallowthehandlingofothersituationstobededuced.

it is important to remember that prpSc is entirely resistant to many standard disin-fection protocols.

3. referenCeSeu.2000.Directive2000/54/ECof theEuropeanParliamentandof theCouncilof18September

2000ontheprotectionofworkersfromrisksrelatedtoexposuretobiologicalagentsatwork

(seventhindividualdirectivewithinthemeaningofArticle16(1)ofDirective89/391/EEC)

who.2003.Laboratorybiosafetymanual.Secondedition(Revised).ReferencenumberWHO/CDS/

CSR/LYO/2003.4.Geneva

Swiss expert Committee for Biosafety.2006.www.efbs.ch

41

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methodology

Bovine Spongiform enCephalopathieS diagnoStiC methodology

1. BSe diagnoSiS at the tSe referenCe laBoratory, BerneThe NeuroCenter at the Veterinary Faculty of the University of Berne is the SwissNationalReferenceLaboratoryforTSEsinanimals.TheNeuroCentermainlyfocusesonBSE,andisregisteredasanofficialBSEReferenceLaboratorybytheWorldOrganisa-tionforAnimalHealth(OIE).Inthisrole,itisthereforeresponsibleforthediagnosisofBSEatboththenationalandinternationallevelsandregularlyconfirmsthediagnosisofsuspectedcasesofBSEfromcountriesotherthanSwitzerland.TheNeuroCenterisalsoresponsiblefortheevaluationofnewBSEtests.

TheBSEReferenceLaboratoryusestwotypesof tests forBSEdiagnosis.Thefirstgroup of tests includes histopathology and immunohistochemistry (IHC). For thesetests, formalin-fixedbrainsectionsareembedded inparaffinand, forhistopathology,stainedwithhaematoxylinandeosin(H&E).ThesectionscanthenbeexaminedforthepresenceofBSE-specificlesions.ForIHC,theabnormalprionproteinassociatedwithBSE(PrPSc)islabelledwithaspecificantibodytoimprovediagnosticspecificity.Optimalbrainfixationandtissueprocessingaretimeconsumingprocedures,andaminimumoftendaysisrequiredtoobtaingoodIHCresults.Anadvantageofthesetestsisthatotherneurologicaldiseasescanalsobedetected.Moredetailedinformationonhistopathol-ogyandIHCisgiveninsection3ofthischapter.

Thesecondgroupoftestsaretherapidtests.Fortherapidtests,fresh,non-formalinfixedbrainmaterialisused.Severalrapidtestsareavailable.Moredetailedinformationonrapidtestsisgiveninsection4ofthischapter.

1.1. Confirmation of cases and data gatheringInSwitzerland,eachsuspectedcaseofBSE(identifiedeitherclinicallyorbyscreeningtest)mustbeconfirmedbytheNeuroCenterbeforeit isofficiallyregisteredasaBSEcase.Forall clinically suspectedBSEcases, theunopenedheadof theanimalmustbesent to theBSE reference laboratory for testingasquicklyaspossible topreventpostmortemartefacts.Additionally,inSwitzerland,allemergencyslaughtercattleandfallenstockhavetobescreenedusingarapidtest. Inthesecases,thecaudalbrain-stemisremovedattheslaughterhousebyqualifiedpersonnelandsenttoalaboratoryauthorized toperformBSErapid tests.Allpositiveandun-interpretablesamplesarethenforwardedtotheBSEReferenceLaboratoryforconfirmation.Data,includingthelocationoforigin,theanimalidentification,thedateonwhichtheanimalwassentforslaughter,andthehistoryandclinicalsigns(ifknown)areincludedwiththesamples.Detailedsamplingproceduresaregiveninsection2ofthischapter.

2. Sample ColleCtion2.1. Brain removal (clinical suspects)All animals clinically suspected of having BSE should be killed with an intravenousinjection of a concentrated barbiturate solution, following sedation or by some other

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humanemeans.Optimally,severalrepresentativeareasofthebrainshouldbeexam-ined;thereforethewholeheadoftheanimalshouldberemovedandsenttothelabo-ratory.Atthelaboratory,thebrainshouldberemovedassoonaspossibleforfurthertesting.

Beforehandling thehead,protectiveclothingshouldbeworn topreventanydirectcontactwithpotentially-infectivematerialsorinhalationofaerosolspossiblycontainingtheBSEagent.Standardprotectiveclothingconsistsofthefollowing:

• alongdisposablegownwithlongsleeves;• protectiveglovesincombinationwithextraprotectivesleeves;• protectiveglassesorfaceshieldincombinationwithamouthcover;• dedicatedlaboratorybootsorshoes.

Forremoval,theheadisplacedwiththeventralsurface(i.e.jaw)up.Thesofttissuesthatcouldgetinthewayareremovedwithasharpknife.Forthenextsteps,anelectricsawcanbeused.First,thefrontpartoftheheadisremovedbymakingatransversecutbetweentheincisorsandthepremolars(topreventthesawbladefrombeingdam-agedbytheincisors).Afterwardsthehead(andbrain)issplitlongitudinallyexactlyonthemidline,startingattheforamenmagnum.Atthispointitisimportanttomakesurethatallbrainstructures,especiallythemedullaoblongata,havebeencorrectlysplitintotwo,approximatelyequal,parts.Otherwise,averysharpknifeorsharpscissorscanbeused tocreateapproximatelyequalhalves.Onehalfof thebrain is then immediatelyplacedinacontainerwithalargevolumeof10%bufferedformalin(forhistopathologyandIHC).Theformalinmustbechangedaftersevendaysandfixationdurationisideallytwoweeks.Fixationcanbeacceleratedbyplacingtheformalincontaineronashaker,buttheminimumfixationtimeremainsfivedays.Afterfixation,thewholebrainisslicedintoabout5-mm-thicktransversesectionsandissubjectedanapproximateexamina-tion.Aftermacroscopicalexamination, themedullaoblongata, cerebellum,midbrain,thalamus, parietal/occipital cortex, hippocampus, frontal cortex and basal nuclei areselectedandtrimmed,thenplacedintocassettesforfurtherprocessing.

Theremaininghalfofthebrainisfirstsampledforrapidtestsandthenfrozenat-20°Cor-80°C.

2.2. Brainstem removal (risk animals and routine screening at slaughter)In cases of emergency slaughter, fallen stock or routine screening, only the caudalbrainstem (medulla oblongata) is removed for testing without opening the skull. Thehead is separated from the body between the atlas and the foramen magnum, andthenplacedwiththeventralsurfaceup.Insomeslaughterhousesthepresentationoftheheadmaybedifferent,butinancasesthecaudalendofthebrainstemshouldbevisible through the foramen magnum. A specially designed spoon is used to removethebrainstemthrough the foramen (Figure1).Thespoon is insertedventrally (alongthetopedge,astheheadisrestingupsidedown)betweenthebrainstemandtheduramaterandadvancedapproximately7cmwhilecarefullymovingtotheleftandtherighttoseverthecranialnervesonbothsides.Damagetothebrainstemcanbeavoidedbykeeping the spoon close to the bone. Then, the spoon is bent downwards to cut thebrainstemfromtherestofthebrain.Thespooniskept inadownwardpositionwhilebeinggentlypulledoutoftheskullthroughtheforamenmagnum,bringingthecaudalbrainstemwith it.Theareaof interestwithin thebrainstem, theobex region, is thenavailablefortestingbyhistopathology,IHCandrapidtests.

43

BSe diagnostic

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Themedullaisthensplitlongitudinally(Figure2),andonehalfisputinformalinforhistopathologyand IHCwhile theotherhalf is reservedandsampled for rapid tests.Theobexregionistargetedforsamplingforalltests.Thefreshtissueremainingaftersamplingforrapidtestsisthenfrozenat-20°Cor-80°C.

3. neuropathology and immunohiStoChemiStry 3.1. preparation of the formalin-fixed brainEachoftheeightformalin-fixedbrainareas(orbrainstemremovedfromriskanimalsandroutinescreeninganimals) isplacedinacassette.Thesecassettesareplacedin

FIGURE 1

removal of the medulla oblongata

FIGURE 2

tissue selected for testing for BSe (histopathology and rapid tests), (s), includes the obex region (o)

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44

98% formicacid foronehour, then removedand replaced in formalin for3-4hours.Afterthistimethecassettesareembeddedinparaffin.Theembeddedbrainsamplesaresliced into4-5µmthicksectionsandplacedonglassslides.ThesesectionsareusedforneuropathologyandIHC.

3.2. neuropathologyFor neuropathology, sections are stained with standard H & E stain using standardprocedures.

TheneuropathologyofTSEhasfivecharacteristicfeatures:1.Nomacroscopic(gross)changesorlesionsarepresent.Therefore,itisnotpos-

sibletoanalyseaBSEsamplewithoutamicroscope.2. Spongiformchangesof thebrain tissue.Thesemostlyoccur in thegreymatter

and are bilaterally symmetrical (although this symmetry is normally unrecog-nized,sinceonlyonehalfof thebrain isexamined).Spongiformchangesoccurin several predilection areas, especially the dorsal nucleus of the vagus nerve(Plates1and2),solitarytractnucleus,nucleusofthespinaltractofthetrigeminalnerveandolivarynucleus(Figure3).However,thesearenottheonlyareaswherespongiformchangescanoccur.Theintensityofchangesisvariable,butthereisnocorrelationbetweentheintensityofthechangesandtheappearanceofclinicalsignsinliveanimals.Cattlewithstrongclinicalsignshavebeenfoundtohavenospongiformchangesofthebraintissue,andcattlewithoutanyclinicalsignscouldhaveahighdensityofvacuoles.

3. Neuronalvacuolation.Aswithspongiformchanges,neuronalvacuolationoccursincertainpredilectionareas,includingthedorsalnucleusofthevagusnerveandthevestibularnuclei.However,itcanalsooccuroutsidetheseareas.Thenumberandsizeofvacuolesinaneuronarevariable.Somehaveonlyonelargevacuole,someseveralsmallones,andsomehavecombinationsofboth.Thevacuolesaremostlyempty,andoften there isnoadditionalneuronal change to theaffectedneuron.Itisimportanttorealizethatneuronalvacuolationcanbenormalincer-tainareas,andthatitisnotasignexclusivetoBSE.

plates 1 and 2:

SeverespongiformchangesinthedorsalnucleusofthevagalnerveofaBSE-positivecow.MagnificationofPlate1100X,magnificationofPlate2400X.

4�

BSe diagnostic

methodology

4. Neuronaldegeneration.Neuronaldegenerationcanoccur in thebrainstemandthalamicnuclei.

5. Gliosis (i.e. hyperplasia and/or proliferation of astrocytes and occasionally ofmicroglialcells).Gliosiscaneitherbeassociatedornotassociatedwithspongi-formchangesofthebraintissueandthepresenceofPrPSc.Thereasonforthisisnotknownatpresent.GliosisisnormallymildinBSEcases,butusuallysevereinscrapiecases.Glialfibrillaryacidicprotein(GFAP)stainingmaybeusefultoshowthepresenceofastrogliosis.

differential diagnosis of spongiform encephalopathy in the bovine brain Afteridentificationofthehistopathologicalfeaturespresentinasample,BSEmustbedifferentiatedfromotherneuraldiseasesshowingsimilarlesions.Thename“encepha-lopathy” refers to the fact that thedisease isprimarilydegenerativeand,apart fromgliosis, does not show any inflammatory changes. The term “spongiform” is purelydescriptiveandissometimesusedinterchangeablywithotherterms,suchasvacuola-tion, spongiosis, spongy degeneration or microcavitation. Vacuolation of the neuropilcanbeseeninmanydifferentdiseasesandeveninnormalbrain,sopossiblecausesofspongiformchangesmustbedifferentiated.

Normalvacuolation:Intraneuronalvacuolescanbefoundinclinicallyhealthycattle,mostlyintherednucleusorinthenucleusoftheoculomotornerve;theyarenotassoci-atedwithanyPrPScaccumulationandareconsideredincidentalfindings.

Pathologicalvacuolation:Vacuolescanbeseeninmanydifferenttoxicand/ormeta-bolicdisorders,insomecongenital/inheriteddiseasesandinsomeinfectiousdiseases.

FIGURE 3

transverse section of the obex region of the medulla oblongata: tracts and nuclei important for tSe neuropathology are identified

legend:ts:solitarytractnts(top):nucleusofthesolitarytractnts(centre):nucleusofthespinaltractoftrigeminalnervenX:dorsalnucleusofthevagusnXII:hypoglossalnucleusF.r.:reticularformationol:olivarynuclei

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techniques for

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spongiform

encephalopathies

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Thefollowinglistgivesexamplesofpossibledifferentialdiagnoses(butisnotmeanttobeexhaustive):Toxic/metabolicdisorders: hepaticencephalopathy renalencephalopathy polioencephalomalaciawithcerebrocorticalnecrosis (thia-

minedeficiency,leadpoisoning,waterdeprivation)Inheriteddiseases: congenitalerrorsofaminoacidmetabolism(suchasmaple

syrupurinediseaseandcitrullinemia)Infectiousdiseases: rabies (Classically a non-suppurative polioencephalomy-

elitis and ganglionitis and may cause only very minimalinflammatorychanges incattle.Vacuolarchangesmaybeencountered,notnecessarilyinassociationwiththeinflam-mation. Negri bodies, which in cattle are mostly found inPurkinjecells,arepathognomonicforrabies.)

Vacuolationduetopostmortemartefacts:Vacuolesmaybeseenasaresultofautoly-sis,inadequatefixation,freezingandproblemsintissueprocessing(particularlywhenthesamplesarekeptin70%alcoholformorethan36to48hours).

To diagnose BSE in clinically suspect cases, the whole brain can be examined forhistological lesions.Table1showstheresultsofbrainexaminationofsuchcasesfortheyear1999inSwitzerland,asanexample.

3.3. immunohistochemistryAfterevaluatingthehistopathologyofthesamplesaccordingtothefivecharacteristicfeatures(describedinsection3.2),IHCcanbeusedtoincreasethespecificityofdiagno-sisbydirectlyidentifyingtheaccumulationofprionprotein(PrPSc).Thisisaccomplishedthrough labelling of PrPSc in the sample with specific antibodies. In Plates 3 and 4,

taBle 1. differential diagnoses for clinical BSe suspect cases at the Swiss BSe reference laboratory in 1�� (after confirmation of negative BSe status) by percent of total samples showing various pathological changes. (of 4� BSe suspect samples submitted to the BSe reference laboratory in Berne in 1��, � were BSe positive and 40 were negative.)

pathological change percent

Nomorphologicalchanges(lesions)inbrain 35.2

Listeriosis 21.0

Bovinesporadicmeningoencephalomyelitis 16.7

Brainedema 4.7

Polioencephalomalacia 4.3

Bacterialencephalitis(undeterminedetiology) 3.6

Cerebellaratrophy 3.2

Brainneoplasias 3.2

Systemdegenerations 3.2

Hepatoencephalopathy 1.4

Neuroaxonaldystrophy(Weaversyndrome) 1.0

Gliosis(undeterminedetiology) 1.0

Miscellaneous 1.5

Total(n=40) 100.0

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BSe diagnostic

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IHCstainsofbrainsectionsofthesameBSE-positivecowpresentedinPlates1and2clearlyshowaccumulationofPrPSc,andconfirmthediagnosisprovisionallymadewithhistopathology.HistopathologicexaminationofthebrainsectioninPlate5mightyieldaquestionableresult,especially if thepathologistdidnothavemuchexperiencewithBSEdiagnostics.However,examinationoftheIHCpreparation(Plate6)fromthesamecowallowsadefinitivediagnosisofBSEtobemade.

However, theantibodiesusedareunabletodifferentiatenormalPrPprotein (PrPC)present in the brain cells from abnormal PrPSc. Therefore, the enzyme proteinase Kmust first be used to destroy the PrPC differentially, while leaving the proteinase KresistantcoreofPrPScunaffected.ThisensuresthatanyPrPdetectedwillbePrPSc.Thisrequirementisexplainedfurtherintherapidtestsection(section4)ofthischapter.

Inaddition,astep todemasktheappropriateepitopeof theproteinaseKresistantcoreofPrPScisrequired,otherwisetheconformationoftheproteinpreventstheanti-bodyfrombinding.Demaskingcanbeaccomplishedbydenaturationoftheproteinorbyusingnon-specificproteases.

IntheIHCprotocolusedattheSwissBSEReferenceLaboratory,theantibodyC15Sis

plates 3 and 4:

PrPScdepositioninthedorsalnucleusofthevagalnerveofthesameBSE-positivecowasinPlates1and2.MagnificationofPlate3100X,magnificationofPlate4400X.

plate �:

VeryfewspongiformchangesinthedorsalnucleusofthevagalnerveofaBSE-positivecow;theclearspacesaroundtheneuronsareartefacts.Magnification100X

plate �:

CorrespondingareatoPlate5,stainedbyIHC:cleardepositionofPrPScintheneuropil.Magnification400X

diagnostic

techniques for

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spongiform

encephalopathies

4�

usedforthedetectionofPrPSc.TheIHCprotocolwasoptimizedforthisantibody,whichisapolyclonal rabbitantiserumraisedagainstapeptideof thebovinePrPsequenceGQGGTHGQWNKPS.ThissequenceislocatedneartheN-terminalofthePrPScprotein-aseKresistantcore.ThisantibodycanbeusedforIHCaswellasforELISAtests,andhasspecificityisagainstbovine,felineandovineproteins.Manyotherantibodies,mostlymonoclonal,arenowcommerciallyavailableandcanbeobtainedforusewithvarioustestingprotocols.

ForallIHCanalyses,apositiveandanegativecontrolshouldberuntogetherwiththeBSEsamplestoruleoutanyproceduralerrors.Thecontrolsamplesmustbetreatedinexactly thesamewayas theactualsamples.Allanalysesshouldbeperformed induplicate.

4. rapid BSe teStS Since1997,testshavebeendevelopedtoanalyseBSEsuspectmaterialsrapidly.TheEUandseveralindividualcountrieshaveintensivelyvalidatedthesetests.Whichrapidtestsarelicensedandapprovedinvariouscountriesthroughouttheworldisvariable.TestsapprovedintheEU(asof14June2006)aregiveninTable2.

Allexistingand licensedBSErapid testshaveseveral things incommon.First,alltestsusematerialfromthebrainstem,implyingthatthesetestsarepostmortemtests.Thesamplesmustbetakenfromtheobexregion(describedinsection2ofthischapter)inordertomaximizesensitivityofthetests.Second,alltestsarecurrentlybasedonthesameprinciplesofhomogenization,proteinaseKdigestion (with theexceptionof theIDEXXHerdChekBSEAntigenEIA),anddetection.Althoughtheprinciplesofthesestepsaresimilarbetweentests,therearesignificantdifferencesintheexecution.

performanceAccording to external evaluations (Moynagh and Schimmel, 1999; EU, 2006) the tentests currently approved in the EU (Table 2) all have excellent sensitivity (100%) andspecificity(100%),whenIHCistakenasthereference(goldstandard)method.

taBle 2. BSe post mortem tests approved in the eu (as of June 200�)

name year of producer principle approval

PrionicsCheckWestern 2001 Prionics,Switzerland Immunoblot

Bio-RadTeSeE 2001 Bio-rad,France SandwichELISA

PrionicsCheckLIA 2003 Prionics,Switzerland SandwichELISA

InProCDI-5 2003 InPro,SanFrancisco,USA Conformation dependant immunoassay

CediTectBSE 2006 Cedidiagnostics,Netherlands ELISA

IDEXXHerdChekBSEAntigenTestKit 2006 IDEXX,Maine,USA ELISA

InstitutPourquierSpeed`it 2006 InstitutPourquier,Montpellier,France SandwichELISA

RoboscreenBetaPrionBSEEIA 2006 RoboscreenLeipzig,Germany SandwichELISA

RocheAppliedSciencePrionScreen 2006 Roche,Basel,Switzerland SandwichELISA

PrionicsCheckPrioStrip 2006 Prionics,Switzerland Lateralflow immunoassay

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Special devicesAlthough the required materials are primarily included in the test kits, the presenceofspecialdevicesandequipment inthelaboratory isaprerequisitefortestingforalltests.Notalltestsrequirethesamedevices,andpricedifferencesamongdevicesareconsiderable.

availability of single componentsAlltestsareprimarilysuppliedaskits,withtherequiredmaterialsforconductingacer-tainnumberoftests.However,itislikelythatnotallmaterialswillbeusedatthesamerate,especiallywhenonlylimitedamountsofsamplesareanalysed.Theavailabilityofsinglecomponentsisthenanadvantageandcouldallowareductionincosts,thoughinsomecasessinglecomponentsarenotnecessarilylessexpensivethanthewholekit.Thekitsdifferintheavailabilityofsinglecomponents.

high throughputLaboratories that participate in a BSE surveillance programme will have to analyserelativelylargeamountsofsamples.Inthiscase,itisimportantthatthetestusedhasahighthroughputpotential.Thispotentialcan,forexample,beincreasedbyautomatingasmanystepsaspossibleinthetestprocedure,aseachsteprequiringmanualhan-dlingreducesthethroughputpotential.

low throughputIncontrast toBSEsurveillance laboratories,BSEreference laboratorieshavearela-tivelylowthroughputofsamples.Thenitisimportantthatthetestusedisalsosuitablefor a small amount of samples. The components (e.g. buffer, antibodies) should beavailableorshouldbeabletobepreparedinsmallamounts.

timeAlthoughalltestsdiscussedherearerapidtests,thetimeneededforanalysisdiffersamongthem.Theshorterthetime,thefasterresultscanbereportedtothecustomer.Thisisespeciallyimportantwhennormalslaughteranimalsarebeingtested,ascar-cassesareoftenonlyreleasedfromtheslaughterhouseaftertestresultsarenegative.

handlingIngeneral,teststhathavefewerhandlingstepsareeasiertoperformandhavelowerriskofhumanerror.Automationoftheteststepsreducestheamountofhumanhan-dling. However, the type of handling is important, as some handling steps are morecomplicatedthanothers.

interpretationThelaststepofthetestprocedureistheinterpretationoftheresults.Computerprint-outswithvaluesdesignatedasoverorbelowastatedcutoffvalueareeasytointerpret.Theinterpretationofawesternblot(WB)resultneedsmoreexperience.

ConclusionsAll tests currently approved in the EU are either based on WB or ELISA technology.Althoughtherearedifferencesbetweenthetests,theoverallperformanceiscompara-

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ble.Greatdifferencescanbefoundinthehandlingandtheversatilityforhighandlowthroughputset-ups.

procedure after positive test resultsTheprocedureforhandlingtestpositiveresultsdiffersbetweentheEUandSwitzerland.WithintheEU,initiallyreactivesamplescanberetestedinduplicateusingthesameteststartingfromthehomogenate.Thetestcannotberepeatedstartingfromtheoriginalbrainmaterial,sincethishasalreadybeenprocessedintohomogenate.Ifatleastoneofthetwoduplicateshasavaluehigherthanthesamplecutoff,thesampleisconsid-eredtobepositiveandthesamplewillbesenttothenationalreferencelaboratoryforconfirmation.

In Switzerland, the initial reactive samples are not retested. The initial reactivesamplesaresenttotheNationalReferenceLaboratory,whereconfirmatorytestsareperformed.

new developmentsWorkisconstantlybeingdoneonthedevelopmentofnewrapidtestsbothbycompaniesthatalreadyproviderapidtestsandbynewcompanies.Newtestscanbebasedontherefinementofanestablishedprocedureoronthereplacementofproceduresbycom-pletelynewconcepts.

Allthesenewtestsarestillbasedonpostmortemsamplingastheyusebrainmate-rialfromtheobexregion.Ofcourse,theabilitytodiagnoseBSEantemortemwouldbeahugeadvantage,andmuchresearchisbeingdoneinthisfield.Reportsonpossibleantemortemtestsarepublishedregularly.However,noneof these testshaveso farpassedthevalidationprocess,andanimminentbreakthroughinantemortemtestingisnotforeseen.

�. referenCeSeC, 2006. BSE diagnostic opinions, Scientific advice. http://europa.eu.int/comm/food/food/

biosafety/bse/sci_advice_en.htm.

moynagh J, Schimmel h. 1999. Tests for BSE evaluated. Bovine spongiform encephalopathy.

Nature400(6740),105.

1appendiX

authors and course contributors

�3

appendix 1

authors and course contributors

authorS and CourSe ContriButorS

Catherine Botteron NeuroCenter,UniversityofBerne,Switzerlandmarcus doherr VetSuisseFaculty,UniversityofBerne,Switzerlandanou dreyfus FAO,AnimalProductionandHealthDivision,Rome,ItalyChristine friedli SAFOSO,Berne,Switzerlanddagmar heim SwissFederalVeterinaryOffice,Berne,Switzerlandulrich Kihm SAFOSO,Berne,Switzerlandelizabeth mumford SAFOSO,Berne,Switzerlandfrancesco proscia FAO,AnimalProductionandHealthDivision,Rome,Italymanon Schuppers SAFOSO,Berne,Switzerlandtorsten Seuberlich NeuroCenter,UniversityofBerne,Switzerlandandrew Speedy FAO,AnimalProductionandHealthDivision,Rome,Italymarc vandevelde NeuroCenter,UniversityofBerne,Switzerlandandreas Zurbriggen NeuroCenter,UniversityofBerne,Switzerland

Participants from the partner countries have also contributed significantly to theproductionand translationof thecoursemanuals,and tomanyotheraspectsof thecourses.

2appendiX

related background reading and web links*

*ThesereferencesandWeblinksrefertoallfourCapacityBuildingforSurveillanceand Prevention of BSE and Other Zoonotic Diseases project course manuals.Therefore,alldocumentsandlinksmaynotbeapplicabletothetopicscoveredinthismanual.

*ThesereferencesandWeblinksrefertoallfourCapacityBuildingforSurveillanceand Prevention of BSE and Other Zoonotic Diseases project course manuals.Therefore,alldocumentsandlinksmaynotbeapplicabletothetopicscoveredinthismanual.

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appendix 2

related background reading and web links

related BaCKground reading and weB linKS

tSe pages of selected ministries and other general data sourcesdepartment of environment food and rural affairs. United Kingdom, BSE homepage:

http://www.defra.gov.uk/animalh/bse/index.html

fao. BSEpages:http://www.fao.org/ag/AGAinfo/subjects/en/health/bse/default.html

ministry of agriculture of new Zealand. BSE homepage: http://www.biosecurity.govt.nz/

node/7650

Swiss federal veterinary office. BSE homepage: http://www.bvet.admin.ch/gesundheit_tiere/

01752/01804/02075/index.html?lang=de

tafS.Positionpapers:http://www.tseandfoodsafety.org/startseite.htm

united States department of agriculture. Animal and Plant Health Inspection Service, BSE

homepage:http://www.aphis.usda.gov/lpa/issues/bse/bse.html

who.BSEpages:http://www.who.int/zoonoses/diseases/bse/en/

international standardsoie. Bovine spongiform encephalopathy. Terrestrial Animal Health Code, Chapter 2.3.13. http://

www.oie.int/eng/normes/MCode/en_chapitre_2.3.13.htm

oie. Factors to consider in conducting the bovine spongiform encephalopathy risk assessment

recommendedinchapter2.3.13.TerrestrialAnimalHealthCode,Appendix3.8.5.http://www.oie.

int/eng/normes/MCode/en_chapitre_3.8.5.htm

oie.Surveillanceforbovinespongiformencephalopathy.TerrestrialAnimalHealthCode,Appendix

3.8.4.http://www.oie.int/eng/normes/MCode/en_chapitre_3.8.4.htm

oie. Procedures for the reduction of infectivity of transmissible spongiform encephalopathy

agents.TerrestrialAnimalHealthCode,Appendix3.6.3.http://www.oie.int/eng/normes/MCode/

en_chapitre_3.6.3.htm

oie.1994.AgreementonSanitaryandPhytosanitaryMeasures.FinalActoftheUruguayRound,Article5.http://www.wto.org/english/docs_e/legal_e/15-sps.pdf

BSe cases and risk eC. BSE testing results of member countries of the EU. http://europa.eu.int/comm/food/food/

biosafety/bse/mthly_reps_en.htm

oie.NumberofreportedcasesofBSEworldwide.http://www.oie.int/eng/info/en_esbmonde.htm

oie.ResolutionNo.XXVII,Recognitionofthebovinespongiformencephalopathystatusofmember

countrieshttp://www.oie.int/eng/info/en_statesb.htm#List

SSC. 2002. Opinion on TSE infectivity distribution in ruminant tissues (state of knowledge,

December2001).AdoptedbytheScientificSteeringCommitteeatitsmeetingof10-11January

2002.http://europa.eu.int/comm/food/fs/sc/ssc/out241_en.pdf

SSC.OpinionsoftheScientificSteeringCommitteeoftheEC.http://europa.eu.int/comm/food/fs/

sc/ssc/outcome_en.html

diagnostic

techniques for

transmissible

spongiform

encephalopathies

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measureseu. 2002. Regulation No 1774/2002. Laying down health rules concerning animal by-products

not intended for human consumption. http://europa.eu.int/eur-lex/pri/en/oj/dat/2002/l_273/

l_27320021010en00010095.pdf

european union guidance document for regulation 1��4/2002. http://europa.eu.int/comm/food/

fs/bse/bse48_en.pdf

fao. 2004. Good practices for the meat industry. FAO Animal Production and Health Manual

No. 2. Rome (also available at: ftp://ftp.fao.org/docrep/fao/007/y5454e/y5454e00.pdf).

fao. 2004. protein sources for the animal feed industry. Proceedings of the FAO Expert

ConsultationandWorkshop,Bangkok,29April-3May2002.FAOAnimalProductionandHealth

ProceedingsNo.1.Rome(alsoavailableathttp://www.fao.org/documents/show_cdr.asp?url_)

file=/docrep/007/y5019e/y5019e00.htm

fao. 2007. Management of transmissible spongiform encephalopathies in livestock feeds andfeeding.Coursemanual,ProjectCapacityBuildingforSurveillanceandPreventionofBSEandOtherZoonoticDiseases.Rome

fao. 2007. Management of transmissible spongiform encephalopathies in meat production.Course manual, Project Capacity Building for Surveillance and Prevention of BSE and OtherZoonoticDiseases.Rome

heim d, Kihm u. 2003. Risk management of transmissible spongiform encephalopathies in

Europe.RevScitechOffintEpiz22(1),179-199

heim d, mumford e.2005.ThefutureofBSEfromtheglobalperspective.MeatScience70:555-

562

heim d, murray n.2004.Possibilities tomanage theBSEepidemic:cohortcullingversusherd

culling–experiencesinSwitzerland.In:Prions:achallengeforscience,medicineandthepublichealthsystem,2nded.EdsHFRabaneau,JCinatl,HWDoerr.Karger,Basel,Switzerland.pp

186-192

oie.2005.DiseasesnotifiabletotheOIE.http://www.oie.int/eng/maladies/en_classification.htm

render – the national magazine of rendering. 2004. Rendering 101: Raw material, renderingprocess,andanimalby-products.http://www.rendermagazine.com/August2004/Rendering101.pdf

the BSe inquiry. 2000.Thereport.TheinquiryintoBSEandvariantCJDintheUnitedKingdom,Volume13: Industryprocessesandcontrols,Chapter6,Rendering.http://www.bseinquiry.gov.

uk/report/volume13/chapter6.htm

diagnosticsefSa. 2006.EFSAScientific reportson theevaluationofBSE/TSE tests.http://www.efsa.eu.int/

science/tse_assessments/bse_tse/catindex_de.html

oie. 2005. Bovine spongiform encephalopathy. Manual of diagnostic tests and vaccines forterrestrialanimals,Chapter2.3.13.http://www.oie.int/eng/normes/mmanual/A_00064.htm

Safar Jg, Scott m, monaghan J, deering C, didorenko S, vergara J, Ball h, legname g, leclerc

e, Solforosi l, Serban h, groth d, Burton dr, prusiner SB, williamson ra. 2002.Measuring

prionscausingbovinespongiformencephalopathyorchronicwastingdiseasebyimmunoassays

andtransgenicmice.NatBiotechnol20(11):1147-1150.

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appendix 2

related background reading and web links

SurveillanceCameron ar, Baldock fC. 1998. Two-stage sampling in surveys to substantiate freedom from

disease.PrevVetMed34:19-30

Salman md.2003.AnimalDiseaseSurveillanceandSurveySystems.MethodsandApplications.

IowaStatePress,Ames,Iowa,USA

Scheaffer rl, mendenhall w, ott l.1990.ElementarySurveySampling.DuxburyPress,Belmont

CA.

Clinical examinationBraun u, Kihm u, pusterla n, Schönmann m.1997.Clinicalexaminationofcattlewithsuspected

bovinespongiformencephalopathy(BSE).SchweizArchTierheilk139:35-41(alsoavailableat:

http://www.bse.unizh.ch/english/examination/htmlsklinischer.htm)

human prion diseasesdepartment of health,UnitedKingdom.CJD-homepage:

http://www.dh.gov.uk/PolicyAndGuidance/HealthAndSocialCareTopics/CJD/fs/en

3appendiX

glossary of technical terms and acronyms*

*ThisglossaryreferstoallfourCapacityBuildingforSurveillanceandPreventionofBSEandOtherZoonoticDiseasesprojectcoursemanuals.Therefore,alldocu-mentsandlinksmaynotbeapplicabletothetopicscoveredinthismanual.

�3

appendix 3

glossary of technical terms and acronyms

gloSSary of teChniCal termS and aCronymS

aafCo AssociationofAmericanFeedControlOfficials

ab Antibody

afia AmericanFeedIndustryAssociation

animal by-products Tissues and other materials (including fallen stock) dis-cardedattheslaughterhouse,whichgenerallygotoincin-eration,burialorrendering(dependingonthecountry)

animal waste Animalby-products

ante mortem Before death (generally refers to the period immediatelybeforeslaughter)

ap Apparentprevalence

BaB Bornaftertheban;animalswithBSEthatwerebornafterimplementationofafeedban

BarB Bornafter the realban;animalswithBSE thatwerebornafter implementation of a comprehensive and effectively-enforcedfeedban

BSC Biosafetycabinet

BSe Bovinespongiformencephalopathy

Bl Biosafetylevel

By-pass proteins Proteinsthatarenotdegradedintherumenbutaredigest-edinthesmallintestinetoprovideadditionalaminoacids

CCp Critical Control Point: a step in a production chain that isessential to prevent or eliminate a food safety hazard orreduceittoanacceptablelevelandatwhichacontrolcanbeapplied

Cen EuropanCommitteeforStandardization

CJd Creutzfeldt-JakobDisease

CnS Centralnervoussystem

Combinable crops Thoseabletobeharvestedwithacombine

Contaminants Materialsthatshouldnotbepresentinagivenproduct;e.g.rodents,birds,rodentdroppings,toxinsandmouldarecon-taminantsthatshouldnotbepresentinanylivestockfeed

Control (noun) The state wherein correct procedures are being followedandcriteriaarebeingmet(HACCPcontext)

Control (verb) Totakeallnecessaryactionstoensureandmaintaincom-pliancewithcriteriaestablishedinaHACCP(orothercon-trol)plan(HACCPcontext)

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Core fragment ThepartofPrPScthatisnotdigestedbyproteinaseK(alsocalledPrPRes)

Critical limit Acriterionthatseparatesacceptabilityfromunacceptability(e.g.duringaudits)

Cross contaminants Substancescarriedfromareasormaterialswheretheyarenotprohibitedtoareasormaterialswheretheyareprohib-ited

Cross feeding The feeding of a livestock group with prohibited feedsintendedforanotherlivestockgroup

Cp Crudeprotein

Cwd Chronicwastingdisease.

dna Deoxyribonucleic acid; the genetic material for all livingorganismsexceptbacteria

downer cattle Cattletoosicktowalktoslaughter(definitiondiffersamongcountries)

eC EuropeanCommission

efSa EuropeanFoodSafetyAuthority

eliSa Enzyme-linkedimmunosorbentassay

emergency slaughter Slaughter cattle with clinical signs non-specific for BSE(definitiondiffersamongcountries)

epitope Structuralpartofanantigenthatreactswithantibodies

epitope demasking Processinwhichtheepitopebecomesavailableforantibodybinding(forexample,bydenaturation)

essential amino acids Those that cannot be synthesized and therefore must beprovidedbythefeed/food

eu EuropeanUnion

fallen stock Cattlethatdiedorwerekilledforunknownreasons(defini-tiondiffersamongcountries)

fao FoodandAgricultureOrganizationoftheUnitedNations

fda FoodandDrugAdministration(UnitedStatesofAmerica)

fefaC EuropeanFeedManufacturers’Federation

fifo Firstinfirstout;aproductionconcepttooptimizequality

flushing batches Batchesof feedprocessedortransported in-betweenfeedbatchescontainingprohibitedandnon-prohibitedmaterials,andintendedtoremovetracesofprohibitedmaterialsfromtheequipment

fmd Foot-and-mouthdisease

fn False negatives; truly-diseased animals that test negativeonadiagnostictest

fp Falsepositives;trulynondiseasedanimalsthattestpositiveonadiagnostictest

fSe Felinespongiformencephalopathy;TSEincats,believedtobecausedbyingestionoftheBSEagent.

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appendix 3


gafta GrainandFeedTradeAssociation

gap Goodagriculturalpractices

gBr GeographicalBSEriskassessment

ghp Goodhygienepractices

gmp GoodManufacturingPractices

gmt Goodmicrobiologicaltechnique

greaves Aproteinaceousby-productoftherenderingprocess

gtm GAFTATradersManual

h & e Haematoxylinandeosinstain

haCCp Hazard Analysis and Critical Control Points: a method toidentifyprocessstepswherea lossorsignificantdeviancefromtherequiredproductqualityandsafetycouldoccurifnotargetedcontrolisapplied

haCCp plan Adocumentprepared inaccordancewith theprinciplesofHACCPtoensurecontrolofhazardsthataresignificantforthesegmentoftheproductionunderconsideration

hazard Abiological,chemicalorphysicalagentwiththepotentialtocauseanadversehealtheffect

hazard analysis The process of collecting and evaluating information onhazardsandconditionsleadingtotheirpresencetodecidewhich are significant for the segment of the produc-tion under consideration and therefore which should beaddressedinthecontrol(orHACCP)plan

high quality protein Proteinsourcesthatmatchtherequirementsofaparticularspeciesorproductionclasswell

hplC Highperformanceliquidchromatography

iag EuropeanFeedMicroscopistsworkinggroup

ifif InternationalFeedIndustryFederation

ihC Immunohistochemistry

indigenous BSe case DomesticBSEcase;non-importedBSEcase

m+C Methioninepluscysteine;aminoacidsgenerallyconsideredtogether,becausecysteinecanbederivedfrommethionineinanimals

iSo InternationalOrganizationforStandardization

mammal Ananimalthatlactates;inthiscontext,livestockexcludingaquaticspeciesandpoultry

mBm Meatandbonemeal; thesolidproteinproductof theren-deringprocess

medulla oblongata Caudalportionofthebrainstem

mmBm Mammalianmeatandbonemeal

monitoring Anongoingprocessofspecificanimalhealthdatacollectionoveradefinedperiodoftime

diagnostic

techniques for

transmissible

spongiform

encephalopathies

��

monogastric species Animalswithsimplestomachs(e.g.swine,poultry,horses,humans)

moSS Monitoringandsurveillancesystem

mrm Mechanicallyrecoveredmeat

nirC Nearinfraredcamera

nirm Nearinfraredmicroscopy

nirS Nearinfraredspectrography

notifiable disease Adiseaseforwhichthereisanationallegalrequirementtoreportcasesandsuspectstoanofficialauthority

obex Thepointonthemidlineofthedorsalsurfaceofthemedullaoblongata thatmarks thecaudalangleof the fourthbrainventricle;amarkerfortheregionofthebrainstemwheresomeof thepredilection areas forhistological lesionsandPrPSc deposition in BSE are located (such as the dorsalnucleusofthevagus)

od Opticaldensity

oie WorldOrganizationforAnimalHealth

or Oddsratio

pathogenicity Ability of an organism to invade a host organism and tocausedisease

pCr Polymerasechainreaction

pithing The laceration of central nervous tissue by means of anelongated rod-shaped instrument introduced into the cra-nialcavityofslaughtercattleafterstunning.

pK Proteinase K; a serine proteinase that digests PrPC com-pletelybutPrPSconlypartiallyundercertainconditions

post mortem Afterdeath

prion InfectiousagentcausingTSE

proteolysis Cleavage of a protein by proteases; also referred to as“digestion”

prp Prion protein, encoded by the gene PRNP, expressed bymanycelltypesandmanyorganisms

prpBSe Resistant prion protein associated with bovine spongiformencephalopathy;alsocalledPrPSc

prpC Normalprionproteinfoundineukaryoticcells

prpres Resistantprionproteincore remainingafterproteolysisofPrPScusingproteinaseK

prpSc Resistant prion protein associated with transmissiblespongiformencephalopathies,includingBSE

prpSens Normalprionproteinfoundineukaryoticcells;alsocalledPrPC

pv Predictivevalue

��

appendix 3


rapid test Test systems using immunological assays that detect thepresence of infectious agents in animal tissues or othermaterialswithinhours

rr Relativerisk

ruminant species Animals with multichambered stomachs that allow bacte-rial fermentationof feedsprior to intestinaldigestion (e.g.cattle,sheep,goats,camellids)

Scrapie ATSEofsheepandgoats

Se Sensitivityofadiagnostictest

Segregation Undesirable separation of raw ingredients in a compoundfeedafterprocessing

Sft SwissInstituteofFeedTechnology

Sick slaughter Cattle with non-specific signs (definition differs amongcountries)

Sp Specificityofadiagnostictest

SpS agreement AgreementontheApplicationofSanitaryandPhytosanitaryMeasures

Srm Specifiedriskmaterials;thoseanimaltissuesmostlikelytocontainTSEinfectivematerial

SSC Scientific Steering Committee of the European Commis-sion

Strip test Lateralflowimmunochromatographictestforrapiddetec-tionofproteinsinfeedsamples

Surveillance Extension of monitoring in which control or eradicationactionistakenonceapredefinedlevelofthehealth-relatedeventhasbeenreached

tafS InternationalForumforTSEandFoodSafety

tBt agreement AgreementonTechnicalBarrierstoTrade

terrestrial animal In thiscontextall livestockexcludingaquaticspecies (e.g.poultry,ruminants,pigs,horses)

tme Transmissibleminkencephalopathy

tp Trueprevalence

tracing Determiningwhereananimalorproductoriginatedorhasbeen

tracking Following an animal or product forward through the sys-tem

tSe Transmissiblespongiformencephalopathy

uK UnitedKingdomofGreatBritainandNorthernIreland

uSa UnitedStatesofAmerica

vCJd Variant (or new variant) Creutzfeldt-Jakob disease ofhumans; believed to be caused by ingestion of the BSEagent

4appendiX

project summary

�1

appendix 4

project summary

proJeCt Summary

ThiscourseisapartoftheprojectCapacityBuildingforSurveillanceandPreventionofBSEandOtherZoonoticDiseases.Theaimoftheprojectistobuildcapacity,establishpreventivemeasuresandanalyserisksforbovinespongiformencephalopathy(BSE),sothat,ultimately,partnercountriesareableeithertoprovethemselvestobeBSE-freeorareabletodecreasetheirBSErisktoanacceptablelevel.Governmentalandprivateveterinary services, diagnostic laboratories, and the livestock, food and animal feedindustrieswillbestrengthenedandsupported,andtechnicalcapacitybuiltateverystepalongthefoodproductionchain.Inthefuture,theknowledgegainedduringthisprojectcould be used by the countries to establish similar programmes for control of otherzoonoticfood-bornepathogens.

TheprojectisfundedbySwissgovernmentalagenciesandutilizesexpertiseavailableinSwitzerlandandworldwideandinfrastructureavailablefromtheFoodandAgricul-tureOrganizationoftheUnitedNations(FAO)toassistthegovernmentsofthepartnercountriestoachievetheproject’saim.TheexecutingagencyisSafeFoodSolutionsInc.(SAFOSO)ofBerne,Switzerland.

The direct project partner in each country is the National Veterinary Office. Thecountriescommitandpayasalary toat leastone individual,situated in theNationalVeterinaryOffice,toactasaNationalProjectCoordinator(NPC),committhreetraineespercourseandprovidethenecessaryinfrastructureforimplementationoftheprojectinthecountry.TheNPCisresponsibleforcoordinatingtheactivitiesoftheprojectwithinthecountry,includingofferingtrainingcourses,identifyingandorganizingtrainees,andpromotingcommunicationbetweentheproject,thegovernment,thescientificcommu-nityinthecountry,thelivestockandfoodindustries,andthepublic.Othercommitmentsby the countries include providing paid leave time for employees to attend courses,providing infrastructure and facilities for in-country courses, providing historical andcurrentdata(surveillancedata,animalmovementdata,import/exportrecords)andthestaffrequiredtoidentifythosedata,andprovidingadequatestaffforandfacilitatingtheinitialneedsassessmentandfinalcomprehensiveriskassessment.

ANationalProjectBoardineachoftheparticipatingcountriesregularlyevaluatestheoperationalprogressandneedsoftheproject,andprovidesaregularvenueforcom-munication among the project team, national partners and stakeholders. This BoardiscomprisedoftheNPC,representativesofthenationalgovernment,aprojectrepre-sentative,thelocalFAOrepresentative,andlocalstakeholdersfromprivateindustryandtheveterinarycommunity.

aCtivitieS of the proJeCt1. Thespecificneedsofeachparticipatingcountryareassessed.2. Comprehensive courses to “train the trainers” are provided in Switzerland (or

elsewhere)toselectedparticipantstoimproveunderstandingoftheepidemiologyofandrelevantriskfactorsforBSEandtodevelopspecificknowledgeandskillsforimplementingappropriatecontrols.

diagnostic

techniques for

transmissible

spongiform

encephalopathies

�2

Threetraineesfromeachcountry,aswellastheNPC,traveltoSwitzerland(orelse-where)toparticipateineachcourse.

Thecoursesare:• DiagnosticTechniquesforTransmissibleSpongiformEncephalopathies• Epidemiology, Surveillance and Risk Assessment for Transmissible Spongiform

Encephalopathies• ManagementofTransmissibleSpongiformEncephalopathiesinLivestockFeeds

andFeeding• Managementoftransmissiblespongiformencephalopathiesinmeatproduction

EachcourseisprecededbyanintroductiontoBSEcoveringthebackgroundoftrans-missiblespongiformencephalopathies,BSE,biosafety,generalconceptsofepidemiolo-gyandriskassessment,andriskcommunication.Eachcoursealsoincludesdiscussionofaspectsofriskcommunicationthatarerelevanttothetopicbeingpresented.

Onlythosemotivatedindividualswhowillbeimplementingtherelevantinformationinto the national BSE programme, who have some experience (e.g. ability to use amicroscope,veterinarytraining)andhaveadequateEnglishskills,areaccepted.

After each course, the relative success of the course is evaluated focusing on thesuccess of the training methods and effectiveness of the knowledge transfer ratherthanonthelearningoftheindividualtrainees.Therefore,nowrittentestisgiven,butclosecontact ismaintainedwiththetraineesaftertheyreturntotheircountries,andtheirprogressandsuccess in implementationof their training into thenationalBSEprogrammeisfollowedandevaluatedinthefield.

3. Eachof theTSE-specificcourses is thenofferedasan in-countrycourse in thenative language, and is organized by the trainees and the National VeterinaryOfficeswithtechnicalsupportfromtheproject.In-countrycoursesusethesamecurriculumandexpectedoutcomesastheoriginalcourses,andareprovidedwithsupport,technicalassistanceandmaterials(translatedintotheirownlanguage).TheintroductoryTSEandbiosafetycoursecurriculumisalsopresented.Atleastoneexpert trainerassists inpresenting thesecourses.Participantsarechosenaccordingtostrictselectioncriteria,butthenumberofparticipantsandthefre-quencyandlocationofcoursesgivendependsontheneedsofthecountryandthetypeofcourse.

4. The knowledge gained through the courses should then be integrated by thepartnercountrythroughdevelopmentandimplementationofanationalBSEcon-trolprogramme.Theprogrammeispromotedandsupportedbythecountriestoensurethesustainabilityofthesystem.Contact,technicalsupportandfollow-upwiththecountriesisongoingthroughouttheproject.

5. InformationcampaignstoimproveBSEawarenessaretargetedtonationalgov-ernments,producersandconsumers.

6. PartnercountriesaresupportedinthesubmissionofacomprehensivenationalBSEriskassessmenttotheWorldOrganisationforAnimalHealth(OIE)inordertodocumenttheirBSEstatustotheinternationalcommunity.

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