New developments in classical microscopy; what can be expected for the official control ·...
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BA SE Biotechnol. Agron. Soc. Environ. 2011 15(S1), 15-24 New developments in classical microscopy; what can be expected for the official control? Leo van Raamsdonk (1) , Luciano Pinotti (2) , Pascal Veys (3) , Monique Bremer (1) , Wilma Hekman (1) , Anniek Kemmers (1) , Anna Campagnoli (2) , Claudia Paltanin (2) , Camino Belinchón Crespo (3) , Jef Vliege (1) , Victor Pinckaers (1) , Jan Sten Jørgensen (4) (1) RIKILT - Institute of Food Safety. Wageningen UR. Bornsesteeg, 45. PO Box 230. NL-6700AE Wageningen (The Netherlands). E-mail: [email protected] (2) University of Milan. Department of Veterinary Sciences and Technology for Food Safety - VSA. Via Trentacoste, 2. I-20134 Milano (Italy). (3) Walloon Agricultural Research Centre (CRA-W). European Union Reference Laboratory for Animal Proteins in Feedingstuffs. Chaussée de Namur, 24. B-5030 Gembloux (Belgium). (4) Danish Plant Directorate. Skovbrynet, 20. DK-2800 Lyngby (Denmark). The official control of animal proteins in feed is focused on the prevention of Bovine Spongiform Encephalopathy (mad cow disease). The current legislation of the European Union is planned to avoid the feeding of animal by-products to the same species as its origin (ban of cannibalism, or species-to-species ban). With respect to the official control, the circumscription of the term species in legislation should be defined, and species-specific markers should be available. Markers will include primer sets, antibodies, near-infrared profiles or visual characteristics. The method of classical light microscopy is currently the only accepted method in the framework of the official detection of animal proteins. Besides the necessary development of complementary methods, either as stand alone methods or in combination, the visual characteristics used for a microscopic examination of meat and bone meal particles should be fully explored. Multivariate analysis of a range of characteristics of lacunae in bone fragments revealed that discrimination is possible between mammalian and avian bone fragments. Translation to features for every day practical use should be carried out very carefully, and only comprehensively collected information on a range of features will give a first indication of the source. Characteristics of hairs and feather filaments can be used to identify the origin of animal particles. An in situ identification method has been developed for antibody conjugation with troponin I in muscle fibers on a microscopic slide. A proof of principle is presented. Interlaboratory transferability and validation have still to be achieved. The development and testing of light microscopy markers in the framework of the SAFEED-PAP project revealed that a fine tuning of existing microscopic characteristics appears to be possible. Keywords. Microscopic method, feed, marker, hair, feather, bone, meat and bone meal. 1. IntroductIon Bovine Spongiform Encephalopathy (mad cow disease) is generally believed to be caused by contamination of animal feeds containing animal by-products contaminated with prions (Prince et al., 2003). Therefore, an impressive set of regulations on processing, storage, incineration, and use in the feed production chain is in force. The microscopic analysis of feed samples for the detection of animal materials such as bone fragments, muscle fibers a.o. is applied from the beginning of the regulations (Directive 98/88/ EC and successors). In the last years a range of other methods for detection, identification and confirmation are developed (latest overview in Fumière et al., 2009; see other contributions in this volume). Nevertheless, light microscopy remains until now as the only one method officially accepted for detection of animal proteins by the European Commission. Regulation 152/2009/EC provides the most recent overview of officially recognized methods for the official control of feed. The strengths of the microscopic detection method are, among others, sufficient detection at contamination levels as low as 0.02% (Engling et al., 2000; van Raamsdonk et al., 2009), the indication of the type of the detected materials, and the insensibility for the sterilization temperature. Animal materials, both fully or partly prohibited, such as meat meal, meat and bone meal, feather meal and fish meal can easily be distinguished from legally applied ingredients, e.g. milk powder, blood meal and gelatin. Weaknesses are the poor abilities to identify the species origin of the materials found, and the need to have skilled laboratory analysts, both for applying the method correctly, and
Transcript of New developments in classical microscopy; what can be expected for the official control ·...
BASE Biotechnol. Agron. Soc. Environ.201115(S1),15-24
Newdevelopmentsinclassicalmicroscopy;whatcanbeexpectedfortheofficialcontrol?LeovanRaamsdonk(1),LucianoPinotti(2),PascalVeys(3),MoniqueBremer(1),WilmaHekman(1),AnniekKemmers(1),AnnaCampagnoli(2),ClaudiaPaltanin(2),CaminoBelinchónCrespo(3),JefVliege(1),VictorPinckaers(1),JanStenJørgensen(4)(1)RIKILT-InstituteofFoodSafety.WageningenUR.Bornsesteeg,45.POBox230.NL-6700AEWageningen(TheNetherlands).E-mail:[email protected](2)UniversityofMilan.DepartmentofVeterinarySciencesandTechnologyforFoodSafety-VSA.ViaTrentacoste,2.I-20134Milano(Italy).(3)WalloonAgriculturalResearchCentre(CRA-W).EuropeanUnionReferenceLaboratoryforAnimalProteinsinFeedingstuffs.ChausséedeNamur,24.B-5030Gembloux(Belgium).(4)DanishPlantDirectorate.Skovbrynet,20.DK-2800Lyngby(Denmark).
TheofficialcontrolofanimalproteinsinfeedisfocusedonthepreventionofBovineSpongiformEncephalopathy(madcowdisease).ThecurrentlegislationoftheEuropeanUnionisplannedtoavoidthefeedingofanimalby-productstothesamespeciesasitsorigin(banofcannibalism,orspecies-to-speciesban).Withrespecttotheofficialcontrol,thecircumscriptionofthetermspeciesinlegislationshouldbedefined,andspecies-specificmarkersshouldbeavailable.Markerswillincludeprimersets,antibodies,near-infraredprofilesorvisualcharacteristics.Themethodofclassicallightmicroscopyiscurrentlytheonlyacceptedmethodintheframeworkoftheofficialdetectionofanimalproteins.Besidesthenecessarydevelopmentofcomplementarymethods,eitherasstandalonemethodsorincombination,thevisualcharacteristicsusedforamicroscopicexaminationofmeatandbonemealparticlesshouldbefullyexplored.Multivariateanalysisofarangeofcharacteristicsoflacunaeinbonefragmentsrevealedthatdiscriminationispossiblebetweenmammalianandavianbonefragments.Translationtofeaturesforeverydaypracticaluseshouldbecarriedoutverycarefully,andonlycomprehensivelycollectedinformationonarangeoffeatureswillgiveafirstindicationofthesource.Characteristicsofhairsandfeatherfilamentscanbeusedtoidentifytheoriginofanimalparticles.Anin situidentificationmethodhasbeendevelopedforantibodyconjugationwithtroponinIinmusclefibersonamicroscopicslide.Aproofofprincipleispresented.Interlaboratorytransferabilityandvalidationhavestilltobeachieved.ThedevelopmentandtestingoflightmicroscopymarkersintheframeworkoftheSAFEED-PAPprojectrevealedthatafinetuningofexistingmicroscopiccharacteristicsappearstobepossible.Keywords.Microscopicmethod,feed,marker,hair,feather,bone,meatandbonemeal.
1. IntroductIon
Bovine Spongiform Encephalopathy (mad cowdisease) is generally believed to be caused bycontamination of animal feeds containing animalby-products contaminated with prions (Prince et al.,2003).Therefore,animpressivesetofregulationsonprocessing, storage, incineration, anduse in the feedproductionchainisinforce.Themicroscopicanalysisoffeedsamplesfor thedetectionofanimalmaterialssuchasbone fragments,musclefibersa.o. isappliedfromthebeginningoftheregulations(Directive98/88/ECandsuccessors).Inthelastyearsarangeofothermethodsfordetection,identificationandconfirmationaredeveloped(latestoverviewinFumièreetal.,2009;seeothercontributionsinthisvolume).Nevertheless,light microscopy remains until now as the only one
method officially accepted for detection of animalproteins by the European Commission. Regulation152/2009/EC provides the most recent overview ofofficially recognizedmethods for the official controlof feed. The strengths of the microscopic detectionmethod are, among others, sufficient detection atcontaminationlevelsaslowas0.02%(Englingetal.,2000;vanRaamsdonketal.,2009), the indicationofthetypeofthedetectedmaterials,andtheinsensibilityfor the sterilization temperature. Animal materials,bothfullyorpartlyprohibited,suchasmeatmeal,meatandbonemeal,feathermealandfishmealcaneasilybedistinguishedfromlegallyappliedingredients,e.g.milkpowder,bloodmealandgelatin.Weaknessesarethepoorabilities to identify thespeciesoriginof thematerialsfound,andtheneedtohaveskilledlaboratoryanalysts,both forapplying themethodcorrectly,and
16 Biotechnol. Agron. Soc. Environ. 201115(S1),15-24 vanRaamsdonckL.,PinottiL.,VeysP.etal.
for a reliable distinction between the allowed andprohibitedingredients.
The European project Stratfeed was started todevelop methods and markers for identificationof animal proteins. A further development of themicroscopicmethodanditsmarkerswasincludedinthe followingSAFEED-PAPproject. In thispaperabriefoverviewofthefurtherdevelopmentsintheareaofmicroscopyofthesemarkersisreported.
2. BacKground
There is no legal limit for meat and bone meal(MBM) in feed. In all cases where animal proteinfromacertainsourceisprohibited,anulltoleranceisapplied.ArangeofDirectivesandRegulationsapplytotheuseorprohibitionofanimalproteins.
All animal proteins are prohibited for feedingto ruminants by TSE Regulation 999/2001/EC.This is the permanent ban. Feeding of by-productsto the same species as the source is prohibited bythe Animal By-Product Regulation 1774/2002/EC(article22:Banofcannibalism,orspecies-to-speciesban).Feedingofmealprocessedfromcaughtfishisallowed for feeding farmedfish, even ifmaterial oftheownspeciesmightbe included.This species-to-speciesban ismeant tobepermanent,but currently“hidden” behind theExtendedFeed ban.Accordingto this Extended feed ban (Regulation 1234/2003/EC) all animal proteins from farmed animals areprohibitedforfeedingtofarmedanimalsagain.Thisquite severe measure is a compromise as long asanimal specific identification methods are not fullydevelopedandvalidated.ThecompletebanonfeedinganimalproteinstoruminantsisrelaxedbyRegulation956/2008/ECwhichallowsthefeedingoffishmealtounweanedfarmedruminants.
Amoredetailedviewonthelegislationpointsoutthatthefeedingofanimalproteins,includingthoseofruminantsource,isallowedforpetsandfuranimals.However, theexemptionsarementionedindifferentRegulations:– pets (dogs, cats, etc.): Regulation 999/2001/EC, article7;– furanimals:Regulation1234/2003/EC,Amendment on Annex IV of Regulation 999/2001/EC; Regulation1774/2002/EC,article22.
Furthermore derogations exist for a rangeof materials such as blood meal, blood plasma,hydrolyzed proteins, gelatin, milk products, eggproducts, tricalciumfosfate. These by-products arelisted in Regulation 999/2001/EC Annex IV, lastamendedbyRegulation1292/2005/ECandRegulation956/2008/EC.
Insignificant amounts of bone fragments aretoleratediningredientsofavegetativesourceintendedfor feeding according to Regulation 163/2009/EC,provided that a risk assessment indicates that a lowrisk level applies.This derogationwas intended foraccepting the presence of bone fragments in rootandtubercrops,possiblyoriginatingfromrodentsorrelatedanimals.
A principal problem is the lack of a definitionof the term “species”. Besides eternal discussionsamong taxonomists concerning species concepts,the legislation is not clear on the definition of theterm“species”.AbiologicalspeciesisindicatedbyaLatinbinomen,suchasSus scrofaforwildboar,andbythenameSus scrofa domesticusfordomesticatedpig, which is an indication of subspecific rank. Ifthis species circumscription is applied to the term“species” in legislation, then Sus scrofa would beallowedtoconsumeby-productsofe.g.Sus barbatus(bearded pig), one of the other pig species. In allcases the term “species” in legislation might pointto a group of species, but it is not specified whattaxonomicgroupingthisshouldbe.
In practice it is assumed to mean that pigsmight consume non-ruminant/non-pig and avianmaterial, and poultry might consume mammalianmaterial, to name the two most prominent non-ruminant farmed animals. The term “ruminant”might either apply to the official orderRuminantia(table 1) or to the group of ruminating animals,which includes camels, llamas and alpacas aswell. In the latter case, identification markers fordiscriminating “ruminants” inwide sense frompigsis very difficult.The explanation of the ban is alsoless clear for horses, turkeys, ostrich, reptiles, etc.Someofthesespeciesaregettingincreasinginterestinfarmingactivities.
Some examples can be given to illustrate thedifficultyofdistinguishingspeciesgroupsforavoidingunintentionalcontaminationintheframeworkofthespecies-to-species ban. Regulation 163/2009/ECanticipatesonthesituationthatremainsofmammalslivingonproductionfieldscanunintentionallyshowup inplantmaterial in the formof “bone spicules”.No indication of species concerned is given inthe Regulation. This could include mice species,rat and vole (belonging to the rodents) as well asrabbit,hareandmole,whichbelongtootherspeciesgroups (table 1).A comparably undefined situationappliestocontaminationsoffishmealbyremainsofsea mammals. Whales, dolphins, seals and relatedanimalsbelongtodifferentgroups.Thedevelopmentofmarkers for identification should be based on anestablishedviewonanimalgroupdefinitions.
Inthefollowingparagraphsmicroscopicmarkerswill be discussed according to their ability to
Newdevelopmentsinclassicalmicroscopy 17
distinguish between groups of animals at differentclassificationlevels.
3. recognItIon of Bone fragments
The microscopic method comprises of severalsteps. After grinding the sample with mesh size of
2mm, an amount of preferably 10g is mixed intetrachloroethylene (TCE). Most ingredients such asplant materials, hair filaments, feather filaments andmuscle fibers remain floating. Only minerals, boneparticles,teethfragmentsandfishscaleswilltogetherformthesediment.Usuallythesedimentcomprisesof100-300mg,dependingontheamountofmineralsinthe original feed. The investigation of the relatively
table 1.Summarizedoverviewof theclassificationof themajor farmedanimals, theirwildrelatives,andanimalsusedas food source.Onlymajor classification levels arementioned, and only species are namedwith relevance to farmingpractices,eitherasfarmedordomesticatedanimal,orashuntedorcaughtsourceofanimalproteins,oraspossiblesourceofunintentionalcontaminationinpartiesofby-productsoffarmedanimals.
class order suborder family representativesdomesticated/farmed Wild
Mammals Even-toedungulates Ruminants Cattle,sheep,goat Deer,elkSuina Pig SwineTylopods Camel
Odd-toedungulates Horse,donkeyWhales Whales,dolphinCarnivores Feliformia Cat
Caniformia Dog,furanimals Sealion,seal,walrusLagomorpha Rabbit Rabbit,hareRodents Rat,mouse,etc.Soricomorpha Mole
Birds Galliformes Poultry,turkey PartridgeAnseriformes Geese,duckColumbiformes PigeonCharadriiformes SeagullStruthioniformes Ostrich
Reptiles Crocodile
Bone(ray-)fish Salmoniformes Salmon Salmon,troutClupeiformes Herring,sardineGadiformes Cod,haddockPleuronectiformes Sole,turbotPerciformes Scombroidei Tuna,mackerel
Percoidei Whiting
Cartilaginousfish Sharks,rays
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smallamountofmaterialofthesedimentstillrepresentstheoriginal10gofsamplematerial.Thesedimentationprocedureistobeconsideredasaconcentrationstepofafactor20orhigher.Inaddition,thefloatingmaterialandapartoftheoriginalsamplecanbeinvestigated.FurtherdetailsonthemicroscopicmethodcanbefoundinRegulation152/2009/EC,andinGizzietal.(2003),vanRaamsdonketal.(2007)andARIES(2010).
Based in the above brief description of themicroscopicmethod,bone fragmentsare theprimarytargetofmicroscopicexamination. It iscommonandlegalpractice todistinguisheasilybetweenmaterialsfromthesuperclassofbonefishandthesuperclassoftetrapods(inthesenseofterrestrialanimals).
Several different markers are being used forthe characterization of bone fragments (e.g. shape,size and density of lacunae, visibility of connectingcanals).One of the first publications on this topic isfrom Pinotti etal. (2004). A range of 32characterspertainingtothelacunaeisexaminedfurtherbyusingimageanalysistechniques.Theanalyseswerebasedonmeasurementsof30individuallacunae(13originatingfrom4mammalsamples,17from4poultrysamples).Themajorcharactersofthevariationbetweenmammalandpoultrymaterialaretheareapolygon(areacoveredby a single lacuna) and the perimeter (length of thelacuna outline). For 28lacunae (93.3%) a correctidentificationwasmade, in twooccasions(6.7%) thelacunaefrompoultrybonefragmentswereincorrectlyclassifiedasbeingmammalian(Pinottietal.,2004).
In the framework of the SAFEED-PAP research863lacunae were measured using both manual andautomatic methods in reference samples containingpoultry and mammalian meat meal and bone meal(Pinottietal.,2008;Campagnolietal.,2009).Inthiscase26charactersweredeterminedonthelacunae,ofthese23differedsignificantly(P<0.001,ANOVA)betweenmammalianandpoultrybone.Their results indicatedthatgradualdifferencesexistbetweenmammalianandpoultrybone characteristics.Box-plots ofmeans andmediansofarangeofvariablesindicatedthatabsolutediscriminationbetweendifferentspeciesgroupsisnotpossiblewhenbasedonsingleparameters,mainlyduetotheoverlapinthedatasets.Combinationsofvariablesmightgivefurtherpossibilitiesfordiscrimination.
In a further study an even more extended setof lacunae measurements was established withdata on 1,143lacunae of 25different samples. The56characterswere ranged ingroupswith correlationcoefficientsof±0.85orhigher.Onlyoneparameterofeachgroupwaschosenforthefinalanalyses,inordertoavoidtoomuchredundancyinthedataset.MultivariateanalysisintheformofaPrincipalComponentAnalysis(PCA)wasappliedtotheresultingdatasetwitheightcharacters for every lacuna. This way of examininga bone fragmentwith a number of lacunae does not
reflect the way in practice to describe the overallviewofabonefragment.Usuallyamicroscopistwillexamineandevaluate thebone fragmentasawhole,reflectingona rangeofdifferent features.Therefore,a new dataset was constructed consisting of eightaverages for thevariablesofeachof the25samples.TheresultofthePCAonthis25x8datasetisshowninfigure 1.Itappearsthatacombinationofcharacterscan differentiate in general betweenmammalian andpoultrybone fragments.Themaindivisionalong thex-axisispredominantlysupportedbythreecharacters:thetotalareacoveredbyalacuna,thewidthofalacuna,and the smoothness of the border of a lacuna. In allcases“lacuna”meanstheaveragerepresentationofthelacunaeinabonefragment,sincethePCAwasbasedonadatasetwithaverages.Thenextveryimportantstepistotranslatetheseresultstotheeverydaylaboratorypractice for providing useful markers for giving atleastafirstindicationaboutthenatureofthefragmentsfound.Thistranslationshouldbecarriedoutcarefully.
ThespatialdistributionoflacunaeinabonefragmentwasanalyzedinSAFEED-PAPusingaseconddataset.Firstindicationsofthisdatasetrevealthatalsointhesecases only gradual differences between mammalianandavianmaterialexist.
As a summarizing first indication, mammalianbone fragments might show larger and relativelywider lacunae, with amore erratic border comparedto avian bone fragments. The lower smoothness ofthe border of lacunae inmammalian bone fragmentsmight be related to the situation that on average thesmall canaliculae, connecting the lacunae, are morevisible. The connections of the canaliculae with thelacunae are visible as irregularities.Amore detailedpresentation and discussion of the results will bepublishedseparately.
4. HaIr and featHer fIlaments
The simple presence of hairs or of feather filamentspoints to an identification at the level of classes(mammals vs birds).More detailed analysis of hairswasobtainedbytheCRA-WintheframeworkoftheSAFEED-PAP project and under the request of theEuropeanCommission.Avisualstudyof“rodent”(seebackgroundinthebeginningofthisarticle),ruminantandpighairswas carriedout showingdifferences atlevel of orders. Furthermore species identification ofRodentiaspeciesispossible.
In normal practice hairs are usually not detectedin samples in regularmonitoringprograms (personalcommunications).Ifsomesmallfragmentsofhairsoroffeathersareanywaypresentinmeatandbonemeals(MBM),thedetectionisdifficult;adedicatedreagentfordetectionofhairsandfeathersisnotroutinelyapplied.
Newdevelopmentsinclassicalmicroscopy 19
ThislowoccurrencemightbeduetoalowabundanceinEUproducedMBM, as an effect of theEuropeanrenderingprocess(133°C,3atmduring20min).TheAnnexofCommissionRegulation242/2010/ECstatesthat“Theproductmustbesubstantiallyfreeofhooves,horn,bristle,hairandfeathers,aswellasdigestivetractcontent.”Presenceoflowamountsofanimalproteinsin feeds can, however, be due to the situation thatoccasionallyrodentsentertheproductionfacilitiesandtheproductflow.Inthesecaseshairscanbeexpectedaswellandtheycanbeusedtodiscriminatebetweenthese unintentional side effects and the presence ofprocessedanimalproteinsinthesenseoftheEuropeanlegislation.
Itisknownthatthemajorgroupsofmammals(i.e.ungulates including ruminants, carnivores includingfur animals and pets, and rodents in wide sense,table 1)canbedistinguishedusinghaircharacteristics(Brunneretal.,1974;Teerink,1991).Intheoccasions
that a feed sample in practice contains one or a fewparticles of animal origin, it would be an advantagetodiscriminate at least between ruminant and rodentmaterialifhairscanbeidentified(figure 2).Inthecaseofanegative result for the identificationof ruminant
figure 1.Plotof thefirst(x-axis)andsecond(y-axis)principalcomponentofadatasetof25animalproteinsamplesand8characteristics.Onlythethreecharacterswiththehighestfactorloadingsareshown.
50µm 50µm 50µm
figure 2. Longitudinal views of hair fragments of cattle(left)andofarodent(centre).Theoutercuticleofahairisshownright(picturesgivenbycourtesyofCRL-AP).
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material,aconfirmedpresenceof rodentsmightbeacomplementary result, explaining the source of theanimal proteins present in the feed. In the case of apresence of ruminant material, the finding of other,includingrodent,material indicates thepresenceofamixtureofanimalmaterials.Thegeneralprincipleofidentifying the origin of animal constituents, also inthe case that these particlesmight not be prohibited,is included in the global analytical scheme ofFumièreetal.(2009).
Staining of a part of the sieve fractions of thewhole feed samplewith cystin reagent, in order toenhancethevisibilityofkeratinasmajorcomponentofhairs,isonlyafacultativestepintheprocedureasdescribedinRegulation152/2009/EC.Thedifferenttypesofhairsasindicatedinliteraturealthoughrare,canevenbefoundafterheattreatment.
Haircharacteristicsanddocumentationhavebeencollected in the framework of SAFEED-PAP. Theresults obtained by SAFEED-PAP partner CRA-Wwill be used as part of the expert systemAnimalRemainsandIdentificationSystem(ARIES).
5. comBInatIon of metHods
Combination of methods can be achieved in orderto join the strengths of several methods, whereasthedisadvantagescanbeminimalised.Thedifferentdetection and identification methods such as PCR(DNAdetection),immunoassays(proteindetection),near-infrared microscopy and light microscopycan be combined in various ways. One possibilityfor combining the strengths of different methodssequentially is the application of a method foridentificationafterapositivedetectionisachievedbyaninitial(screening)method.OneexampleistorunPCRonasediment,indicatedaspositiveafterlightmicroscopy(Toyodaetal.,2004;Fumièreetal.,2006).Itisalsopossibletocombinetwoapproachesinonemethod. In situdetectionorhybridization isknownfor years as a powerful method for detection andidentificationofsmallquantitiesandsmallparticles(Jinetal.,1997;Leitchetal.,2004;Harrison,2007).ThecombinationoflightmicroscopyandeitherPCRor immunochemicalanalysisadds thepossibilityofidentifying individualparticles to the achievementsof themicroscopicmethod: its sensitivity to detectanimal proteins at low contamination levels andits specificity. An on the spot identification ofmicroscopicallydetectedparticleswithrespecttothesource(speciesgroup)wouldenhancethesupportofthelegislation,especiallythespecies-to-speciesban(1774/2002/EC).
A method combining light microscopy with anidentification technique (in situ identification) has
been developed in SAFEED-PAP. Muscles werechosen as primary target, because this is a wellrecognizabletypeofparticlesinanimalproteins,andaprimarytargetfortheexaminationofsievefractionsin the microscopy method. Muscle material showsthecombinationofhighabundanceandthepresenceof an identification mark with high specificity(DNA,protein).Both rt-PCRand immunochemicaldetection are suitable for application to musclematerial. Immunochemical techniques were chosenfor the identification, since antibodies are availablefor troponinI as well as other muscle proteins,whereas a second antibody labeled with a stainingenzymeisavailableaswell.IntheframeworkoftheSAFEED-PAPproject severalantibodiesare raised.Anantibodywithaspecificresponseandsensitivityforruminants(cattleandsheep)hasbeenused.Thisantibodyshowsaminorreactiontopigproteins,andnosignalforpoultryandfishmaterials.
The design of a combinationmethod comprisesseveral steps. The chosen target, present at lowfrequenciesifanyisfound,hastobeconcentratedandselectedfromthefeedsample.Asolventisrequiredwitharelatively lowdensity,whichallowstogetaflotationwiththemusclefibers,andasedimentwiththemajorityof theotherparticles.Asecondstep isto immobilize theparticles fromtheconcentrateonamicroscopicslide.ThedriedflotationissprinkledonaslidewhichiscoatedwithNorlandResin81(r),andhardenedwithUVlight inorder to immobilizethe particles. Optimal circumstances have to beestablishedforhybridizationof thefirstandsecondantibody,andfor thestainingprocedure.Therefore,slides are at first blocked with a buffer containingindifferent proteins, and washed at several pointsintheprocedurewithaTRIS-buffer.Itappearsthatseveral enzyme-substrate combinations connectedto the secondary goat-anti-mouse antibody can beused effectively, either with alkaline phosphatase(blue staining; figure 3) or with horse-radishperoxidise(redstaining).Thesesystemscanbeusedsimultaneously,allowingatheoreticaldiscriminationsystemformusclefibersfromdifferenttargetanimalspecies.
It can be assumed that muscle fibers are notevenly sterilized during the rendering process, andthat as a result the susceptibility of the troponinIcomplex is also unevenly distributed. In images ofmuscle fibers fully applied to the first and secondantibody conjugations and the staining procedure,predominantlythesarcomeresarestained(figure 3:left). In the control (without the first antibodyincubationstep)nocolorreactionisfound(figure 3:centre). Since the troponinI complex is foundin the sarcomeres, this result indicates a specificreaction between the proteins and the antibody.
Newdevelopmentsinclassicalmicroscopy 21
Theresultafterapplying thesameantibodyagainstfish muscle fibers resulted in an irregular stainingpattern(figure 3:right),whichisunderacompoundmicroscope not in focuswith the sarcomeres. Thiscould be indicated as an a-specific color reaction.Thefirst resultsareencouraging,but transferabilityandvalidationofthemethodstillneedtoberealized.More detailed reports on the development of thein situ combination method will be publishedseparately.
6. strategy of control
Several approaches for the control to support thespecies-to-speciesbancanbedesigned(Baetenetal.,2005; van Raamsdonk et al., 2007; Fumière et al.,2009). These approaches should include detection,identificationand,ifrequired,confirmationsteps.Theavailablemethodsshouldbeappliedinoneofthosethree steps according to their strengths. A secondprerequisiteisthatthegroupingofspecies(table 1)will direct the choice for certain identificationmethods and for the use of group-specificmarkers(primersets,antibodies,visualcharacteristics).TheschemeofFumièreetal.(2009)lookscompleteandprecise.Asindicatedintheirpaper,atolerancelevelis currently not part of European legislation, but itisincludedinthescheme.Furthermore,asampleinwhich animal constituents are found can be routedthrough four different investigations including thedetectionmethodleadingto thepositiveresult, twoidentificationstepsandafinalconfirmationmethod.In thispapera simplified investigationschemewillbepresentedwhichincludesthein situidentificationmethod.
Itislikelytoacceptlightmicroscopyasprimarydetectionmethodforitsadvantageslistedinthestartof this paper. Also for the primary discriminationbetweenfishandterrestrialanimalslightmicroscopyis preferred. Reliable identification at lowertaxonomic levels needs other methods, althougha first indication can already be achieved withmicroscopic characteristics.A range of primer setsfor rt-PCR is readily available (Hormisch, 2004;Brolletal.,2007;Shinodaetal.,2008;Rojasetal.,2009).Thedevelopmentof reliable antibodies asksforarelativelyhighinvestment,butat lower levelsofclassification(e.g.mammalianorders)andforveryquickmethodstheseantibodiesareagoodchoice.
A strategy as presented in figure 4 could beimagined. The application of PCR on sedimentscontainingbonefragmentsavoidslargelytheproblemofpositivesignalsfrommilkandbloodproducts.Thereadily available antibodies for ruminant materialcanbeappliedin situ,givingtheopportunitytohavetheadvantageofavery lowlevelofdetection(onemusclefiber).
Confirmationcouldbenecessaryinselectedcases.MassSpectroscopymethodsareindevelopmentforthispurpose.
7. conclusIon
The development and testing of light microscopymarkers in the framework of the SAFEED-PAPproject revealed that a fine tuning of existingmicroscopic characteristics could be achieved.Visual markers could be applied at severalclassification levels (table 1): the discriminationbetweenfishandterrestrialanimals,afirstindicationofthediscriminationbetweenmammalianandavianmaterial (bone fragments, hairs vs feathers), theidentification of different (groups of) mammalianorders (hair types), and the discrimination betweenruminant vs non-ruminant material (muscle fibers)supported by a specific antibody conjugation andstainingprocedure.
Currently hairs are found in samples frommonitoringprogramsataverylowfrequency.Ithasto be investigated whether these rare occurrencesareduetothesituationthataspecialcolorreactionis normally not applied, or that the frequency ofoccurrence is really low.A further analysis of theapplicabilityofhairidentificationisrecommended.
Thecurrentapplicationofthein situhybridizationmethodisbasedonaruminantantibody.Antibodiesraised against other animal species (groups) couldbe applied as well, resulting in a broader range ofapplication.Investmentsinthedevelopmentofotherantibodiescouldbeveryvaluableifvalidated.
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figure 3.EffectofstainingofmusclefibersbyVectorBluestainingsystem.Left:fiberofcattlewithstainedsarcomeres;centre:fiberof cattlewithunstained sarcomeres (control);right:fiberoffishwithunstainedsarcomeres,andplaquesontheoutermembrane(unspecificreaction).The largearrowpointstoastainedsarcomere,thesmallarrowstounstainedsarcomeres.Scalebaris50μm.
22 Biotechnol. Agron. Soc. Environ. 201115(S1),15-23 vanRaamsdonckL.,PinottiL.,BremerM.etal.
All these strategic possibilities resulting fromtheSAFEED-PAPresearcharebasedonthefurtherdevelopment of markers in the framework of theofficialmethod,whichallowsthemtobeusedunder
Regulation 152/2009/EC. The visual microscopicmethod and the in situ combinationmethod can beusedinabroaderframeworkwithotheridentificationandconfirmationmethods(figure 4).
figure 4.Acontrolstrategyforsupportingthespecies-to-speciesbanwithfocusondetectionandidentification.Aconfirmationmethodcanbeaddedtotheprocedure.ItcanbechosentoapplyPCRtotheflotationinadditiontotheapplicationofthecombinationmethod.Rejectionofasampledependsonthelegalprohibitions.
Sample
Microscopy
Totalban
Sediment?
Flotation
PCRCombinationmethod
Ruminant
Otheridentification
method
Rejected Approved
ProhibitedPAPYes No
Yes
No
Yes
No
No
Yes
Pos. Neg.
Newdevelopmentsinclassicalmicroscopy 23
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