commiss decis june establish criteria zone offici surveil suspicion confirm presenc infecti salmon anaemia isa notifi document number text eea relev ec commiss european commun regard treati establish european commun regard council direct eec januari anim health condit govern place market aquacultur anim product amend regul ec articl thereof regard council direct ec june introduc minimum commun measur control fish diseas amend commiss decis ec articl articl thereof direct eec establish sampl laboratori test presenc list list ii diseas refer annex direct eec carri method establish accord articl direct eec sampl plan diagnost method detect confirm list ii fish diseas viral haemorrhag septicaemia vh infecti haematopoiet necrosi ihn laid commiss decis ec articl articl direct eec farm situat water catchment area coastal area farm suspect confirm infect infecti salmon anaemia isa viru situat offici surveil criteria zone offici surveil establish order defin sampl plan diagnost method detect confirm isa establish criteria zone offici surveil suspicion confirm isa fish health laboratori expert consult guidelin diagnosi isa laid current edit diagnost manual aquat anim diseas intern offic epizooti oie account suffici period time provid implement requir measur provid decis accord opinion stand committe food chain anim health adopt decis articl sampl plan diagnost method detect confirm infecti salmon anaemia isa criteria zone offici surveil suspicion confirm isa laid annex decis articl decis appli octob articl decis address member state brussel june commiss david byrn member commiss oj oj oj oj oj annex sampl plan diagnost method detect confirm infecti salmon anaemia isa criteria zone offici surveil suspicion confirm isa introduct definit annex guidelin minimum requir sampl plan diagnost method detect confirm presenc isa integr provis definit laid direct eec direct eec set provis aim proper diagnosi control surveil isa case suspicion confirm isa direct author respons control isa laboratori personnel perform test regard diseas emphasi put sampl procedur principl applic laboratori test evalu result detail laboratori techniqu laboratori appli modif test annex test provid equal superior sensit specif demonstr criteria establish zone offici surveil suspicion confirm isa laid purpos annex addit definit appli quot water catchment area quot mean entir catchment area sourc waterwai estuari part catchment area sourc waterwai natur artifici barrier prevent fish migrat barrier quot coastal area quot mean part coast seawat estuari precis geograph delimit consist homogen hydrodynam system seri system part lai gener principl criteria diagnosi confirm isa criteria zone offici surveil carri suspicion confirm isa part ii set inspect sampl carri order detect presenc isa part iii lai method virolog examin part iv outlin procedur examin sampl rt pcr detect isa part describ protocol examin kidnei imprint ifat indirect fluoresc antibodi test regard isa part vi includ methodolog histolog part vii list acronym abbrevi criteria diagnosi isa establish zone control measur offici surveil gener principl diagnosi isa reason ground fish suspect infect isav outlin part annex member state ensur suspicion fish farm infect isav offici investig confirm rule presenc diseas carri quickli appli inspect clinic examin collect select sampl method laboratori examin laid part iii vi annex order offici confirm presenc isa set criteria laid part annex fulfil suspicion infect isa presenc isa suspect criteria met presenc post mortem find consist isa clinic sign diseas post mortem find clinic sign diseas accord laid current edit oie diagnost manual aquat anim diseas isol identif isav cell cultur singl sampl fish farm part iii reason evid presenc isav independ laboratori test rt pcr part iv ifat part transfer live fish farm reason ground suspect isa present time fish transfer investig reveal substanti epidemiolog link isa suspect confirm farm suspicion isa rule continu investig involv clinic inspect month period month reveal signific evid presenc isa confirm isa presenc isa consid confirm criteria met clinic sign post mortem find consist isa accord current edit oie diagnost manual aquat anim diseas includ dead weak abnorm behav fish sign anaemia post mortem find patholog observ isav detect method isol identif isav cell cultur sampl fish farm part iii ii detect isav mean rt pcr method part iv iii detect isav tissu tissu prepar mean specif antibodi isav ifat kidnei imprint part isol identif isav sampl fish farm test separ occas method part iii isol identif isav sampl fish farm method part iii corrobor evid isav tissu prepar fish farm rt pcr part iv ifat part criteria establish revoc zone control offici surveil suspicion confirm isa purpos establish risk base offici surveil programm member state vicin farm offici suspect confirm infect isa establish control surveil zone zone establish defin base case case analysi risk spread diseas accord epizootiolog situat water catchment area coastal area concern defin control zone extens water catchment coastal area divid control zone surveil zone prevent spread isa compromis addit surveil zone establish water catchment coastal area main factor establish zone influenc risk spread diseas farm wild fish number rate distribut mortal fish farm suspect confirm infect isav mortal farm concern distanc densiti neighbour farm contact farm speci present farm manag appli affect neighbour farm hydrodynam condit factor epidemiolog signific identifi framework epizoot investig carri accord articl direct eec minimum criteria appli establish zone quot control zone quot establish member state close vicin farm confirm infect isav coastal area area includ circl radiu tidal excurs km centr confirm isav infect farm equival area determin hydrodynam epidemiolog data inland area entir water catchment area farm confirm isav infect member state extens water catchment area limit extens zone part water catchment area provid prevent spread isa compromis quot temporari control zone quot establish case suspicion presenc isa base criteria outlin control zone quot surveil zone quot establish member state control zone area intens surveil deem suffici correspond coastal area area surround control zone overlap tidal excurs zone area surround control zone includ circl radiu km centr control zone equival area determin hydrodynam epidemiolog data inland area need extend area establish control zone fallow revoc zone establish compet author member state ensur farm control zone subject period fallow empti fish disinfect durat fallow period farm confirm infect isa month length fallow period farm control zone determin compet author case case risk evalu farm control zone empti week synchronis fallow appli compet author decid fallow farm establish surveil zone establish control zone revok restock farm situat zone empti fish disinfect fallow accord restock zone carri control zone convert surveil zone laid establish temporari control zone revok suspicion isa rule accord part case confirm isa accord part temporari control zone convert control zone establish surveil zone revok year revoc control zone offici surveil suspicion confirm isa refer articl direct eec order establish distribut evolut diseas suspicion confirm isa farm risk base offici surveil programm carri compet author qualifi fish health servic consult control compet author farm situat zone establish purpos applic offici surveil programm compet author inspect spot identifi farm establish zone make offici censu speci categori number fish farm includ mortal figur initi offici censu farm temporari establish control zone keep atlant salmon salmo salar speci refer recent edit oie aquat anim health code suscept potenti carrier isa report mortal dai compet author increas mortal report dai cage compet author investig significantli increas mortal farm suspicion confirm farm establish control zone report weekli mortal cage dai basi compet author farm surveil zone report mortal compet author dai inspect carri regularli year establish zone frequenc outlin tabl climat condit make inspect imposs part year member state lai inspect frequenc conting plan tabl offici surveil programm gt tabl gt surveil programm carri zone revok inspect select collect prepar shipment sampl carri defin part ii ii examin sampl accord part iii vi ii inspect sampl ii inspect select collect sampl farm presenc isa suspect ii regular inspect carri framework offici surveil programm outlin part farm suspect infect isa farm facil cage tank pond inspect presenc dead weak abnorm behav fish recent mortal decompos weak abnorm behav fish examin clinic sign post mortem find isa current edit oie diagnost manual aquat anim diseas ii recent clinic sign consist isa observ inspector veterinarian reason suspect fish infect minimum fish sampl sampl made recent mortal weak abnorm behav fish insuffici number clinic affect fish number sampl supplement healthi fish select cage tank pond show highest number mortal fish show clinic sign diseas ii recent mortal weak abnorm behav fish observ clinic sign post mortem find consist isa sampl obligatori sampl requir carri differenti diagnosi inspector veterinarian discret ii fish wild suspect infect isa member state ensur sampl examin clinic laboratori method laid part ii vi order rule confirm presenc isa estim occurr diseas present signific threat farm fish ii prepar sampl fish ii sampl histolog examin freshli kill fish exhibit clinic sign post mortem find consist presenc diseas extern intern lesion sampl case sampl liver mid kidnei heart spleen remov individu fish scalpel transfer vol vol buffer formol salin ratio fix tissu ensur satisfactori preserv tissu ii tissu virolog examin fish sampl duplic sampl corrobor purpos piec liver anterior kidnei heart spleen remov fish steril instrument transfer plastic tube ml transport solut cell cultur medium antibiot combin μg ml fungizon iu ml polymixin μg ml kanamycin suitabl combin proven effici tissu fish collect tube transport solut repres pool sampl weight tissu sampl ii kidnei imprint ifat examin freshli kill fish hour death piec mid kidnei remov fish steril instrument tissu blot absorb paper remov excess blood repeatedli press poli lysin coat glass slide individu impress adjac overlap give continu singl layer cell blood tissu fluid relev materi test leav kidnei sampl quot drain quot absorb paper avoid lead blood clot caus larg amount serum protein deposit test slide imprint air dri cool dry fix immedi fixat imprint carri hour fish sampl altern imprint frozen air dry store month prior fixat ii fish show sign anaemia stun heparinis blood sampl immedi haematolog examin measur haematocrit ii tissu rt pcr analysi fish sampl piec anterior mid kidnei remov fish steril instrument transfer microfug tube ml rna preserv solut proven efficaci tissu fish collect tube preserv solut repres pool sampl weight tissu sampl approxim fish small obtain sampl requir weight piec kidnei heart spleen liver pylor caeca order prefer make ii shipment sampl fish ii blood sampl tube fish tissu virolog examin rt pcr analysi insul contain instanc thick wall polystyren box suffici ic quot freez block quot ensur chill sampl transport laboratori freez avoid ic present transport box receipt quot freez block quot partli complet frozen except circumst rt pcr sampl sampl virolog examin snap frozen transport laboratori ii slide ifat ship slide holder suffici desicc imprint dry chill ii fish tissu transport fix histolog examin ship leak proof tube impact resist contain thick wall polystyren box ii sampl frozen virolog examin start hour collect sampl sampl corrobor analysi store arriv laboratori ii fish transport laboratori temperatur requir transport ii fulfil fish wrap absorb paper ship plastic bag chill mention ii live fish ship supervis offici servic ii rt pcr analysi tissu preserv rnalat rna extract carri time sampl store temperatur time gt tabl gt ii packag label perform accord present nation intern transport regul ii collect supplementari diagnost materi agreement diagnost laboratori fish tissu collect prepar supplementari examin iii virolog examin iii prepar sampl iii practic difficulti aris make imposs inocul cell hour collect tissu sampl accept freez tissu dai tissu frozen thaw examin iii sampl tissu pool transport solut complet homogenis stomach blender mortar pestl centrifug minut supernat filter μm incub equal volum suitabl dilut pool antisera indigen serotyp ipnv titr antiserum plaqu neutralis test mixtur incub hour repres inoculum treatment inocula antiserum ipn viru viru part europ occur fish sampl aim prevent cpe due ipn viru develop inocul cell cultur reduc durat virolog examin number case occurr cpe consid potenti indic isav sampl product unit consid free ipn treatment inocula antiserum ipn viru omit iii inocul cell cultur iii shk cell pass lower cell grown medium foetal bovin serum mm glutamin mm mercaptoethanol plate cell line proven effect sensit isol isav take consider strain variabl abil strain replic cell line antiserum treat organ suspens inocul young activ grow cell cultur give final dilut tissu materi cultur medium organ suspens μl inoculum ad ml cultur medium minimis risk cross contamin recommend separ plate sampl fish farm site iii plate left uninocul serv neg control separ plate inocul refer isol isav posit control hundr μl stock prepar isav minimum titr tcid ml inocul mix volum materi transfer make dilut mix repeat plate make fold dilut stock isav store year thaw dai note care prevent cross contamin test plate posit control materi avoid risk posit control set handl separ test plate iii sampl incub dai iii microscopi microscop cell cultur examin cpe dai inocul pool show cpe viru identif procedur initi immedi iii cpe observ dai haemadsorpt test iii perform iii haemadsorpt replic isav cell cultur result cpe subject haemadsorpt test altern subject test iii iii cell cultur medium remov includ posit neg control label steril tube hundr μl suspens wash rabbit hors red blood cell suspens wash rainbow trout atlant salmon red blood cell ad incub room temperatur minut red blood cell remov wash medium examin microscop iii presenc cluster red blood cell attach surfac shk cell indic presumpt infect orthomyxoviru haemadsorpt test posit viru identif test perform immedi iii iii subcultiv passag iii subcultiv carri dai hundr twenti μl cultur supernat ad well fresh activ grow shk cell plate incub dai microscop cell cultur examin cpe dai inocul pool show cpe viru identif procedur initi immedi iii cpe observ dai haemadsorpt test perform iii iii cytotox occur dai incub subcultiv perform stage cell incub dai subcultiv dai incub cytotox occur dai subcultiv perform cell incub achiev total dai incub primari inocul iii bacteri contamin occur primari cultur test set tissu homogen store prior inocul tissu homogen centrifug minut supernat filter μm bacteri contamin occur subcultiv step supernat filter μm inocul fresh cell incub dai iii viru identif test evid cpe observ stage haemadsorpt test posit viru identif carri method choic identif isav iii rt pcr part iv consid virus present recommend supplementari viru identif test carri test allow definit identif viru week supernat forward nation refer laboratori eu refer laboratori fish diseas identif iii iii shk cell pass lower cell grown medium foetal bovin serum mm glutamin mm mercaptoethanol plate greater confluenc cell line growth medium proven efficaci hundr twenti μl put viru infect cultur supernat ad well mix μl transfer well dilut addit well left uninocul act control sampl fish farm site handl separ plate viru control viru control establish refer isol isav iii plate incub examin microscop dai earli cpe observ cpe observ dai step fixat well wash pb fix incub aceton minut room temperatur plate air dri stain immedi store hour prior stain iii replic well stain monoclon antibodi isav monoclon antibodi proven effect specif dilut pb incub minut monoclon antibodi remov plate wash time tween pb anti mous igg fitc conjug dilut pb ad incub minut note dilut batch monoclon antibodi fitc conjug optimis laboratori antibodi remov plate wash time tween pb iii well examin immedi invert microscop set fluoresc microscopi suitabl filter excit fitc test consid posit fluoresc cell observ test valid posit control score posit neg control score neg iv examin sampl rt pcr iv section describ procedur requir pcr amplif part segment isav genom carri fish tissu isav cultur iv rna extract rnalat remov sampl ml depc treat dh ad tube tube centrifug rpm minut supernat remov sampl μl trizol invitrogen altern reagent proven equal greater efficaci ad sampl control tube suitabl control materi μl dh kidnei homogen pathogen free fish tissu disrupt repeat pipet tube incub room temperatur minut μl chloroform ad tube tube shaken vigor minut centrifug rpm minut aqueou upper layer remov label ml microfug tube μl isopropanol tube incub minut room temperatur centrifug rpm minut supernat remov ml ethanol ad rna pellet tube centrifug rpm minut supernat remov tube left open approxim minut remain ethanol evapor μl depc treat dh ad resuspend pellet vortex briefli spectrophotomet calcul rna concentr puriti sampl optic densiti measur nm rna immedi dai temporarili store rna store iv rt μg rna dilut depc treat dh ml microfug tube rna concentr sampl low μg rt reaction maximum amount rna dilut rna incub minut tube rna ic rt reagent ad give final concentr buffer mm dntp ng random hexam rnase inhibitor mmlv rt total volum μl tube incub hour cdna store requir pcr iv pcr μl cdna ad μl pcr mix give final concentr buffer mm mgcl mm dntp pmol primer taq polymeras primer isa ggc tat cta cca tga acg aat forward primer isa gcc aag tgt aag tag cac tcc revers primer neg control extract rt pcr step includ tube thermocycl program minut cycl minut minut minut final incub minut result pcr assess electrophoresi agaros gel stain ethidium bromid includ size marker alongsid sampl neg control rt pcr step singl pcr product bp consid indic presenc isav rna sampl addit product bp consid isav rna sampl yield multipl pcr product includ approxim bp isav rna investig dna probe nucleotid sequenc iv pcr confirm isol isav tissu cultur full cpe occur virolog examin tissu sampl shk cell μl supernat remov steril ml tube rna extract sampl iii rt pcr carri cultur full cpe supernat remov cell scrape surfac flask steril ml tube rna extract rt pcr iv dna probe confirm pcr product specif bp pcr product assess probe oligonucleotid hybridis region pcr product intern primer pcr product subject electrophoresi agaros gel alongsid size marker posit control neg control rt pcr step dna southern blot membran label oligonucleotid cgggagttgatcagacatgcactga aggtg incub membran pre hybridis step unbound specif bound probe wash membran bound probe visualis probe bind fragment bp bp present evid specif pcr isav rna present sampl iv nucleotid sequenc pcr product specif pcr assess examin nucleotid sequenc bp pcr product pcr product purifi agaros gel solut fragment sequenc primer pcr vector primer clone vector prior sequenc nucleotid sequenc compar isav segment embl nucleotid sequenc databas access number aj aj presenc sequenc isav segment evid sampl contain isav rna examin kidnei imprint ifat protocol establish examin kidnei imprint ifat prepar stain imprint slide fix aceton methanol aceton minut air dri stain slide examin region slide circumscrib immedgetm pen similar allow air dry slide block solut skim milk pb tween incub gentl agit minut room temperatur slide drain horizont slide box wet tissu paper maintain humid atmospher imprint cover solut monoclon antibodi isav antibodi proven specif efficaci slide box close incub agit minut room temperatur antibodi dilut skim milk actual dilut determin batch slide wash time minut pb tween imprint cover solut fitc goat anti mous conjug dilut skim milk incub humid environ minut room temperatur slide wash time minut pb tween slide cover citifluortm solut ul citifluortm mix ml tween pb suitabl mount medium minut slide wash time pb tween counter stain requir imprint cover propidium iodid mg ml pb tween incub minut room temperatur slide wash time minut pb tween slide drain mount citifluortm suitabl mount medium slide store dark prior microscop examin examin fluoresc microscopi slide examin microscop suitabl epi fluoresc illumin suitabl filter excit fitc caus emit characterist green fluoresc field region defin immedgetm pen examin object suspici area show green fluoresc examin object phase fluoresc illumin ensur fluoresc stain cell stage coordin suspici region record confirm natur fluoresc examin examin primari reader slide posit suspici examin reader result confirm control type control includ batch slide stain ifat kidnei imprint uninfect atlant salmon neg control uninfect shk cell cultur suscept cell cultur neg control isav infect shk cell cultur suscept cell cultur posit control kidnei imprint isav infect atlant salmon recommend addit posit control posit result obtain neg control test consid invalid slide batch slide batch includ posit control neg test consid invalid slide batch case failur control invalid batch slide slide destroi test carri duplic imprint examin tissu techniqu appli fish tissu liver spleen heart provid reason quantiti endotheli cell leucocyt lymphocyt deposit slide stain procedur remain tissu tissu prefer omit propidium iodid stain reli phase illumin identifi cell type present imprint vi histolog paraffin embed section cut μm stain haematoxylin eosin histolog isa current edit oie diagnost manual aquat anim diseas vii acronym abbrevi gt tabl gt