commiss decis februari lai sampl plan diagnost method detect confirm fish diseas repeal decis eec 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 direct ec articl thereof sampl plan diagnost method detect confirm fish diseas laid commis decis eec amend commiss decis ec time adopt decis eec practic scientif develop place direct eec amend requir sampl plan diagnost method updat updat relat examin identif virus caus viral haemorrhag septicaemia vh infecti haematopoiet necrosi ihn accord amend direct eec commun refer laboratori fish diseas establish council direct eec consult sampl plan diagnost method detect confirm fish diseas introduc decis eec repeal sake clariti measur provid decis accord opinion stand veterinari committe adopt decis articl sampl plan diagnost method detect confirm viral haemorrhag septicaemia vh infecti haematopoiet necrosi ihn laid annex articl present decis repeal decis eec articl decis address member state brussel februari commiss david byrn member commiss oj oj oj oj oj annex sampl plan diagnost method detect confirm viral haemorrhag septicaemia vh infecti haematopoiet necrosi ihn introduct annex guidelin minimum requir sampl plan diagnost method detect confirm presenc viral haemorrhag septicaemia vh infecti haematopoiet necrosi ihn integr provis annex direct eec approv mainten statu zone farm approv zone set provis aim proper diagnosi vh ihn offici recognit statu zone farm approv zone accord articl direct eec direct author respons control vh ihn laboratori personnel perform test diseas emphasi put sampl procedur principl applic laboratori test evalu result detail laboratori techniqu laboratori appli modif test annex test provid equal sensit specif demonstr part includ sampl plan diagnost method surveil vh ihn order obtain maintain approv statu zone farm approv zone part ii describ diagnost procedur confirm vh ihn event suspicion part iii set criteria guidelin offici health inspect programm document histor freedom vh ihn part iv recommend procedur vh ihn viru titrat verifi suscept cell cultur infect acronym abbrevi list part part sampl plan diagnost method vh ihn surveil order obtain maintain approv statu zone farm approv zone inspect sampl gener provis clinic health inspect collect select sampl surveil zone farm approv zone order achiev maintain approv statu vh ihn clinic health inspect sampl fish tissu ovarian fluid carri zone farm approv zone order achiev maintain approv statu vh ihn accord annex direct eec summaris tabl detail laid part tabl applic farm farm recomm activ fish egg gamet approv zone approv farm approv zone provid compli requir laid direct eec annex part ii ii clinic inspect carri period octob june water temperatur farm clinic inspect year interv inspect month product unit pond tank net cage inspect presenc dead weak abnorm behav fish attent paid water outlet area weak fish tend accumul water current fish sampl select rainbow trout present fish speci select sampl rainbow trout present sampl obtain fish speci present speci suscept vhsv ihnv list annex direct eec speci proportion repres sampl water sourc utilis fish product fish repres water sourc includ sampl weak abnorm behav freshli dead decompos fish present primarili fish select fish present fish select includ appear healthi fish collect part farm year class proportion repres sampl specif provis includ collect sampl surveil zone farm approv zone order achiev maintain approv statu vh ihn zone farm approv zone supervis offici servic obtain approv statu vh ihn subject model year surveil programm year absenc clinic sign vh ihn farm zone farm approv zone approv health inspect year year year control period preced achiev approv statu absenc clinic sign vh ihn continu sampl collect examin accord tabl sampl select prepar examin part iv laboratori examin produc neg result vh ihn model year surveil programm reduc sampl size offici health inspect programm document histor freedom vh ihn year farm zone farm approv zone approv health inspect year year year control period preced achiev approv statu absenc clinic sign vh ihn continu sampl collect examin accord tabl sampl select prepar examin part iv laboratori examin produc neg result vh ihn order health inspect programm recognis offici servic document freedom vh ihn fulfil criteria guidelin set part iii special provis approv farm farm recomm activ fish egg gamet approv zone approv farm approv zone farm farm recomm activ fish egg gamet approv zone approv farm approv zone obtain statu accord requir laid direct eec annex ii sampl provis laid model part applic farm surveil programm mainten approv statu vh ihn order maintain approv statu zone farm approv zone vh ihn inspect sampl examin carri accord tabl sampl select prepar examin part iv laboratori examin neg agent vh ihn prepar shipment sampl fish shipment transfer laboratori piec organ examin remov fish steril dissect tool transfer steril plastic tube transport medium cell cultur medium calf serum antibiot combin iu penicillin μg streptomycin μg kanamycin millilitr ml recommend antibiot proven effici tissu materi examin spleen anterior kidnei addit heart encephalon case ovarian fluid examin tabl ovarian fluid organ piec maximum fish tabl collect steril tube ml transport medium repres pool sampl tissu sampl weigh minimum gram tube insul contain instanc thick wall polystyren box suffici ic quot freez block quot ensur chill sampl transport laboratori freez avoid temperatur sampl transit exce ic present transport box receipt freez block partli complet frozen virolog examin start hour collect sampl except case virolog examin start latest hour collect materi provid materi examin protect transport medium temperatur requir transport fulfil part paragraph fish laboratori temperatur requir transport fulfil fish wrap paper absorpt capac final ship plastic bag chill mention live fish ship packag label perform accord present nation intern transport regul collect supplementari diagnost materi agreement involv diagnost laboratori fish tissu collect prepar supplementari examin ii prepar sampl virolog examin freez except case practic difficulti aris bad weather condit work dai laboratori problem make imposs inocul cell hour collect tissu sampl accept freez tissu specimen cell cultur medium carri virolog examin dai tissu frozen thaw examin record detail reason freez tissu sampl storm cell line di homogenis organ laboratori tissu tube complet homogenis stomach blender mortar pestl steril sand subsequ suspend origin transport medium sampl consist fish cm long minc steril scissor scalpel remov bodi gut open sampl consist fish bodi length cm cm viscera includ kidnei collect sampl consist fish cm long tissu specimen collect part tissu specimen minc steril scissor scalpel homogenis suspend transport medium final ratio tissu materi transport medium adjust laboratori centrifug homogen homogen centrifug refriger centrifug minut supernat collect treat hour overnight antibiot gentamicin mg ml stage shipment sampl made transport medium exposur antibiot treatment supernat antibiot omit antibiot treatment aim control bacteri contamin sampl make filtrat membran filter unnecessari collect supernat store hour sampl reus virolog examin practic difficulti aris incub breakdown problem cell cultur make imposs inocul cell hour collect tissu sampl accept freez supernat carri virolog examin dai prior inocul cell supernat mix equal part suitabl dilut pool antisera indigen serotyp ipn viru incub minimum hour maximum hour titr antiserum plaqu neutralis test 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 vhsv ihnv sampl product unit consid free ipn treatment inocula antiserum ipn viru omit iii virolog examin cell cultur media bf rtg epc fhm cell grown suitabl medium eagl mem modif thereof supplement foetal bovin serum antibiot standard concentr cell cultiv close vial recommend buffer medium bicarbon medium cultiv cell open unit buffer tri hcl mm na bicarbon mm ph cell cultur inocul tissu materi young hour activ grow confluent inocul inocul cell cultur antibiot treat organ suspens inocul cell cultur dilut primari dilut addit dilut thereof result final dilut tissu materi cell cultur medium order prevent homolog interfer cell line inocul part iii ratio inoculum size volum cell cultur medium dilut cell line minimum cm cell area cell cultur trai utilis cell cultur trai recommend unit similar bigger growth area accept incub cell cultur inocul cell cultur incub dai colour cell cultur medium red yellow indic medium acidif ph adjust steril bicarbon solut equival substanc perform ensur cell suscept viru infect month decreas cell suscept suspect titrat frozen stock vhsv ihnv perform verifi suscept cell cultur infect recommend procedur present part iv microscopi inocul cell cultur inspect regularli time week occurr cpe magnif obviou cpe observ viru identif procedur part iv initi immedi subcultiv cpe develop primari incub dai subcultiv perform fresh cell cultur utilis cell area similar primari cultur aliquot medium supernat cultur well constitut primari cultur pool cell line dai inocul pool inocul homolog cell cultur undilut dilut result final dilut supernat part iii altern aliquot medium constitut primari cultur inocul directli fresh cell cultur subcultiv inocul preced preincub dilut antiserum ipn viru dilut part ii inocul cultur incub dai observ part iii toxic cpe occur dai incub subcultiv perform stage cell incub dai subcultiv dai incub toxic cpe develop dai cell pass incub achiev total dai primari inocul evid toxic final dai incub bacteri contamin occur treatment antibiot subcultiv preced centrifug min filtrat supernat μm filter low protein bind membran addit subcultiv procedur toxic cpe iv viru identif viru identif test evid cpe observ cell cultur medium supernat collect examin techniqu neutralis elisa test allow definit identif viru week supernat forward nation refer laboratori eu refer laboratori fish diseas identif neutralis remov cell collect supernat centrifug membran filtrat μm low protein bind membran dilut supernat cell cultur medium aliquot supernat dilut mix incub minut equal part reagent separ serum group specif antibodi vhsv vol vol dilut serum group specif antibodi ihnv vol vol dilut pool antisera indigen serotyp ipnv vol vol dilut medium posit control viru supernat serum mixtur cell cultur inocul μl incub develop cpe check part iii vhsv strain react neutralis test isol identifi elisa neutralis test proven effici altern viru isol identifi coverglass equival seed cell densiti lead confluenc hour cultiv epc cell recommend purpos strong adher glass surfac cell line bf rtg fhm cell sediment glass surfac hour seed cultur incub hour viru identifi inocul cultur inocul volum volum ratio cultur ratio incub hour incub cultur rins eagl mem serum fix ic cold aceton stain mean layer ifat reagent layer consist polyclon monoclon antibodi refer qualiti reagent layer fluorochrom conjug antiserum immunoglobulin layer antisera test high dose low dose inocul cultur stain proper neg posit control includ test fluorochrom fitc tritc recommend mount stain cultur glycerol salin examin incid ultraviolet uv light eyepiec object len numer apertur amp gt amp gt techniqu techniqu regard cell cultur fixat antibodi refer qualiti proven effici altern elisa well microtitr plate coat overnight recommend dilut purifi immunoglobulin fraction antibodi refer qualiti rins well pb tween buffer viru identifi ad well fold dilut step allow react coat antibodi minut rins pb tween buffer biotinyl antibodi specif coat antibodi ad allow react minut rins hrp conjug streptavidin ad allow react hour rins bound enzym visualis elisa substrat opd biotin avidin base elisa version elisa version proven effici tabl inspect sampl scheme zone farm approv zone year control period preced achiev approv statu vh ihn accord direct eec annex provis set part annex gt tabl gt maximum number fish pool tabl inspect sampl scheme year control period preced achiev approv statu vh ihn zone farm approv zone offici recognis document histori freedom diseas accord direct eec annex provis set part iii annex gt tabl gt maximum number fish pool tabl inspect sampl scheme zone farm approv zone order maintain approv statu vh ihn accord direct eec annex provis set part annex gt tabl gt maximum number fish pool part ii diagnost procedur confirm vh ihn suspect outbreak techniqu carri diagnosi vh ihn convent viru isol subsequ serolog viru identif viru isol simultan serolog viru identif diagnost techniqu ifat elisa confirm case vh ihn farm approv zone base method method tissu materi meant virolog examin case accompani supplementari materi bacteriolog parasitolog histolog examin differenti diagnosi convent viru isol subsequ serolog viru identif select sampl fish show typic sign ihn vh select examin prepar shipment sampl fish laid part collect supplementari diagnost materi laid part ii prepar sampl virolog examin laid part ii iii virolog examin laid part iii iv viru identif laid part iv viru isol simultan serolog viru identif select sampl laid part ii prepar shipment sampl fish laid part collect supplementari diagnost materi laid part ii homogenis organ laid part ii ii centrifug homogen homogen centrifug refriger centrifug minut supernat collect treat hour antibiot gentamicin mg ml membran filter μm low protein bind membran ii treatment supernat diagnost antisera antibiot treat membran filter organ suspens dilut cell cultur medium aliquot mix incub minut equal part reagent list part iv iii cell cultur media laid part iii iii inocul cell cultur viru serum mixtur prepar laid part ii ii cell cultur cell line inocul μl iii incub cell cultur laid part iii iii microscopi inocul cell cultur inspect daili occurr cpe magnif cpe prevent antisera viru consid identifi cpe prevent antisera viru ne identif procedur part iv perform iii subcultiv cpe occur dai subcultiv perform cultur inocul supernat medium part ii ii part iii diagnost techniqu supernat prepar part ii submit ifat elisa part iv part iv rapid techniqu supplement virolog examin hour sampl neg result obtain posit result obtain materi repres case ihn vh approv zone tissu materi subject diagnost techniqu rt pcr frozen section immunohistochemistri formalin fix tissu materi techniqu accompani inocul fix tissu materi cell cultur part iii document histori freedom vh ihn zone farm approv zone guidelin criteria offici health inspect programm health inspect programm initi offici recognis erad programm vhsv ihnv includ remov fish farm clean disinfect fallow restock fish approv farm fish farm histori vhsv ihnv infect health inspect programm base clinic inspect laboratori examin programm includ annual clinic health inspect guidelin part inspect carri year fish tissu ovarian fluid sampl collect farm sampl select prepar subject laboratori examin part ii iv health inspect programm conduct year farm zone farm approv zone approv offici recognit programm case vh ihn occur detect clinic infect viru isol part iv procedur titrat verifi suscept cell cultur infect recommend procedur titrat refer part liil vhsv isol isol ihnv isol repres major group virus eu vhsv pathogen isol rainbow trout freshwat marin isol pathogen turbot ihnv rainbow trout pathogen strain europ defin isol member state refer isol eu refer laboratori fish diseas batch viru low cell cultur passag number propag cell cultur flask bf rtg cell vhsv epc fhm cell ihnv cell cultur medium serum low moi inocul amp lt total cpe viru harvest centrifug cell cultur supernat minut filter sterilis ìm membran filter distribut label cryotub viru week freez replic vial viru thaw cold water titrat respect cell line month suspect suscept cell line decreas viru isol thaw titrat titrat procedur detail procedur time titrat end point dilut includ replic dilut step titr compar previous obtain titr titr viru isol drop factor log compar initi titr cell line longer surveil purpos cell line laboratori line examin separ record year part acronym abbrevi bf bluegil fry cell line cpe cytopath effect crl commun refer laboratori fish diseas elisa enzym link immunosorb assai epc epithelioma papulosum cyprini cell line fhm fathead minnow cell line fitc fluorescein isothiocyan hepe hydroxyethylpiperazin ethan sulphon acid hrp horseradish peroxidas immunofluoresc ifat indirect fluoresc antibodi test ihn infecti haematopoiet necrosi viru ipn infecti pancreat necrosi viru mem minimum essenti medium moi multipl infect ratio number infecti viru particl ad number cell cultur opd ortho phenylen diamin pb phosphat buffer salin rtg rainbow trout gonad cell line rt pcr revers transcriptas polymeras chain reaction tri hcl tri hydroxymethyl aminomethan hcl tritc tetramethyl rhodamin isothiocyan vh viral haemorrhag septicaemia viru except case fish collect remot area possibl daili mail refer laboratori regard cytotox antisera refer laboratori regard cytotox antisera refer laboratori regard cytotox antisera