ARCHIVES HISTORIQUES
DELACOMMISSION
COLLECTION RELIEEDES
DOCUMENTS "COM"
COM(78)614
Vol.1978/0238

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COMMISSION OFTHEEUROPEAN COMMUNITIES
COM(78)614final
J-JBruiseIs,
V'Aâ'COM(78)614final
-November1978
Proposalfora
COUNCILDIRECTIVE
amendingDirective64/432/EECinrespectofenzootic
leukosisamongcattle
»
(submittedtotheCouncilbytheCommission )
C0M(78)614final

Enzooticbovineleukosis(E.B.L.)isaninfectiousvirusdiseaseinducing
proliferationofleukocytes(whitebloodcells)andcausingabloodcancer.
1-2%ofcowsininfectedherdsarelosteachyearduetothesetumours.
ThediseasehasbeenrecognisedinmostpartsoftheworldincludingEurope
it'sspreadbeingbywayoftradeinliveanimalsfromNorthEasternEurope.
Insomeregions10-20%ofherdshavebeendemonstrated tobeinfected.In
certainMemberStatesE.B.L.isundereradication bynationalcontrolsystems
allbreedingherdsbeingmonitoredbymeansoftests.Surveillance isnow
facilitatedbyamoreefficienttest(serological).OtherMemberStateshave
notyettakendecisionsconcerningeradicationalthoughtheyhaveobservedthe
disease.
WhileCommunityguaranteesconcerningE.B.L.incattletradedbetweenMember
StateshavenotuntilnowbeenprovidedinDirective64/432/EEC,themechanism
ofArticle8.2ofthatDirectivehasbeenavailedofbyfiveMemberStatesto
obtainhealthguaranteeswhenbreedingorproductioncattlearetraded.Itis
nowconsideredessentialthatcommunityrulesbeadoptedanditisproposed
thatMemberStateswhichhavesetupE.B.L.freeherdsshouldbeafforded
protection whenanimalsaretradedintosuchherdsfromotherMemberStates.
Becauseofthefragmentary natureofrules-intheMemberStatesconcerning E.B
atpresentthisproposalshouldbeseenasafirststeptowardsmoreelaborate
controlsystemswhichinthefutureshouldleadtotheeradicationofthe
diseasewithintheCommunity .

■Proposai
CouncilDirective
of
amendingDirective64/432/EECinrespectof
enzooticleukosisamongcattle
THECOUNCIL OFTHEEUROPEAN COMMUNITIES ,
HavingregardtotheTreatyestablishing theEuropeanEconomicCommunity,and
inparticularArticlesA3and100thereof,
HavingregardtotheproposalfromtheCommission,
HavingregardtotheopinionoftheEuropeanParliament,
HavingregardtotheopinionoftheEconomicandSocialCommittee,
'whereasoneofthetasksoftheCommunityintheveterinaryfieldistoimprove
thestateofhealthoflivestock,andthustomakestock-breeding moreprofitable;
WhereasthereisaneedtoprotecttheCommunityagainstenzooticleukosisamong
1 2cattle;whereastheCommunity,byCouncilDirective 77/391/EECand78/52/EEC,has
alreadytakenmeasurestoeradicatethisdisease;
Whereassuchmeasuresmustcontribute totheabolitionofbarrierstotradein
freshmeatorliveanimalsbetweenMemberStates,whichareduetodifferences in
healthsituations;
whereasthemeasuresforprotection againstenzooticleucosisamongcattleshould
accordingly beincorporated inCouncilDirective 64/432/EECof26June1964on
animalhealthproblemsaffectingintra-Community tradeinbovineanimalsandswine^
aslastamendedbyDirective
Whereasprovisionshouldbemadeforcertainspecialtemporarymeasuresinorderto
facilitatetheintroduction ofsuchprotection measures;
Whereas,withregardtoenzooticleucosisamongcattle,theriskofthedisease
spreadingmustbeassessedaccordingtocategoriesofanimals;whereasprovision
shouldthereforebemadeforderogations applicabletoanimalsformeatproduction,
andanimalsforslaughtershouldnotbeincludedinthesystemprovidedfor,
0JNoL145,13.6.1977,p.44.
20JNoL15,19.1.1978,p.34.
30JNo121,29.7.1964,p.1977/64.
40JNo

-2-
HASADOPTED THIS DIRECTIVE :
Article 1
Directive 64/432/EEC isamended asfollows :
1.InArticle 2thefollowing point Cp)isadded :
"(P)Enzootic bovine leukosis free herd :means abovine herd which satisfies
the conditions laid down inAnnex 6.
Article 2
2.InArticle 3thefollowing point (e)isadded toparagraph 3:
toparagraph 3:
Ce)inthecase ofanimals consigned toanenzootic bovine leukosis free
herd ,come from anenzootic bovine leukosis free herd and have reacted
negatively toaserological test carried outduring the 30days before
loading and inaccordance with theprovisions ofAnnex H."

-3-
3.InArticle 7thefollowing point (c)isadded tosub-paragraph Bof
oaragraph 1:
"(c)those animals which ,bywayofderogation from Article 3(3 )(e)donot
come from anenzootic bovine leukosis free herd .However these animals
must come from abovine herd allofwhose animals have reacted negatively
toaserological test carried out during the 30days before loading and
inaccordance with Annex H.
This provision shall apply until 31December 1981 J'
A. Thefollowing Article 7aisinserted :
Article 7a
Countries ofdestination shall grant toexporting countries general
authorisations for the introduction into their territories ofbovine animals
formeat production ,under 30months ofage,which bywayofderogation from
Article 3(3)(e ),donot come from anenzootic bovine leukosis free herd .
Such animals shall atloading fortransportation tothecountry ofdestination
bear aspecial identification mark .The Member States ofdestination shall
take all necessary measures toensure that the animals shall atnotime come
into contact with enzootic bovine leukosis free herds intheir territories.*'

Thefollowing isadded topoint <a)ofAnnex E:
"Enzootic bovine leukosis inall its forms ."
1.Inpoint VoftheHealth Certificate ,Model 1,inAnnex F,there is
inserted after paragraph (d)anewparagraph (d,j)asfollows :
(d,j)-they comefrom anenzootic bovine leukosis freeherd (2)
they donotcome from anenzootic bovine leukosis free herd (2)
The result oftheserological test carried outwithin the
prescribed 30days time limit (5)was negative (2).
-they arebovine animals intended only formeat production and
donot come from abovine herd which isenzootic bovine leukosis
free (2)(11).

2.ThefollowingisaddedtothefootnotestoModel1inAnnexF.
(11)Thisderogation appliesonlyinthecase
ofbovineanimalsformeatproduction onconditionthattheybeara
specialmarkandarespeciallysupervisedinthecountryof
destination .
Article7
"Thefollowing Annexes GandHareadded:
ANNEXG
ENZOOTIC BOVINELEUKOSIS FREEHERD
A.Abovineherdisconsidered tobeenzooticbovineleukosisfreeif:
(a)withinthelastthreeyearsnoleukotictumourcasesorleukotic
infiltrations associated withbovineleukosisvirusinliveor
deadanimalshavebeenofficially detectedandnootherfactshave
beenreportedwhichmightgiverisetosuspicionofenzootic
bovineleukosisintheherd.
(b)allthebovineanimalsover12monthsofagehavereacted
negatively toatleasttwoofficialserological testscarriedout
inaccordance withAnnexHatintervalsofatleast4monthsand
atmost12months.
(c)intheintervalbetweenthefirstandsecondserological tests
describedunderparagraph (b),nobovineanimalhasbeenintro­
ducedintotheherdwithoutacertificate fromanofficialveter­
inariandeclaringthattheanimalcomesfrom,
-anenzooticbovineleukosisfreeherd,
or
-abovineherdinwhichallanimalshavereactednegativelyto
aserologicaltestcarriedoutinaccordancewithAnnexHduring
the30daysbeforeitwastakenintotheherd.
B.Abovineherdconsideredtobeenzooticbovineleukosisfreeaccording
toparagraph Ashallcontinuetobesoconsideredif:

-6-
(a)since the herds approval asenzootic bovine leukosis free no
leukotic tumour cases orleukotic infiltration associated with bovine
leukosis virus inlive ordead animals have been officially detected
and noother facts have been reported which might give rise to
suspicion ofenzootic bovine leukosis inthe herd .
(b)all thebovine animals over 12months ofage have reacted negatively
toserological check-tests carried out inaccordance with Annex H
atintervals ofatleast 12months and atmost 3years after the
approval ofthe herd asenzootic bovine leukosis free .
(c)nobovine animal has been introduced into the herd without a
certificate from anofficial veterinarian declaring that the animal
comes from :
-anenzootic bovine leukosis free herd ,
or
-abovine herd inwhich allanimals have reacted negatively toa
serological test carried out inaccordance with Annex Hduring
the 30days before itwas taken into the herd .
C. (a)Until 31March 1980 andnotwithstanding theprovisions ofArticle
2(p)abovine herd mayalso beconsidered tobeenzootic bovine
leukosis free provided that ,
(i)within the last three years noleukotic tumour cases or
leukotic infiltrations inlive ordead animals have been
officially detected andnoother facts have been reported
which might give rise tosuspicion ofenzootic bovine leukosis
inthe herd .
(ii)before 1January 1979 every animal over twoyears ofage
forming part ofthebovine herd hasbeen thesubject oftwo
blood tests forleukosis atintervals ofatleast 12months
which have given noindication ofanabnormal increase inthe
number oflymphocytes .
(iii)theherd conforms tothenational rules oftheMember State
concerned andisdeemed tobeenzootic bovine leukosis free
according tothose rules on31December 1978 .

-7-
(b)Thebloodtestreferredtoinparagraph (a)(ii)shallcomply
withthefollowingrequirements :
Ci)accountshallbetakenoftheabsolutenumberofleukocytes
andtheproportionoflymphocytes .Thetotalnumberof
lymphocytes percubicmillimetreshallbethedetermining
factorandshallbecalculated accordingtothefollowing
formula :
totalnumberofleukocytes percubicmillimetre x
percentageoflymphocytes
100
(ii)thepresenceofanabnormalincreaseinthenumberof
lymphocytes shallbeconcludedinthelightofthefollowing
data:
forbovineanimals :
ofmorethantwoyearsandnotmorethanthreeyears-more
than10500lymphocytes percubicmillimetre;
ofmorethanthreeyearsandnotmorethanfouryears-more
than9500lymphocytes percubicmillimetre;
ofmorethanfouryearsandnotmorethanfiveyears-more
than8500lymphocytes percubicmillimetre;
ofmorethanfiveyearsandnotmorethansixyears-more
than8000lymphocytes percubicmillimetre;
ofmorethansixyears-morethan7500lymphocytes per
cubicmillimetre;
(iii)iftheresultofthebloodtestreferredtoinparagraph (a)
(ii)indicates arelativelyhighlymphocyte counti.e.a
numberoflymphocytes lessthan2000belowthefiguresmentioned
in(b)(ii),thesamesamplemustbeimmediately givenafresh
test,theresultofwhichshallformthebasisofthefinal
evaluation .

ANNcX H
Aqar gel immuno diffusion test
1.Theantigen tobeused inthetest shall contain bovine leukosis virus
glyco proteins .Theantigen shall bestandardised against astandard
serum (E.I. serum )supplied bytheState Veterinary Serum Laboratory ,
Copenhagen .
2.Theofficial institutes indicated below shall bemade responsible for
calibrating theworking laboratory standard antigen against theofficial
E.E.C. standard serum (E.I. serum )provided bytheState Veterinary Serum
Laboratory -Copenhagen .
(a)Germany Bundesforschungsanstalt fürVirus­
krankheiten der Tiere -Tübingen
(b)Belgium Institut national derecherches
vétérinaires ,Bruxelles
(c)France Laboratoire desMédicaments Vétérinaires ,
Fougères
(d)Grand Duchy ofLuxembourg
(e)Italy Instituto Zooprof ilattico Sperimentale ,
Perugi a
(f)Nether lands Centraal Diergeneeskundig Instituut ,
Afdeling Rotterdam
(g)Denmark Statens Veterinaere Serum Laboratorium ,
Copenhagen
(h)Ireland Veterinary Research Laboratory ,
Abbotstown ,Dublin
(i)United Kingdom The Central Veterinary Laboratory ,
Weybridge ,England .
3.Theworking laboratory standard antigens shall besubmitted fortesting
against theofficial E.E.C. standard serum atleast once ayear tothe
E.E.C. reference laboratories listed inparagraph 2above .Besides this
standardisation theantigen inuse canbecalibrated according to
paragraph B.
4.The reagents forthe test shall consist of :
a)antigen :theantigen shall contain specific glycoproteins of
enzootic bovine leukosis virus which has been standardised
against the official E.E.C. serum .

-9-
b)thetestsérum
*
e)knownpositive control serum
d)Agar-gel
0.8%agar
8.5%NaCl
0.05MTris-buffer pH7.2
15mlofthisagarshallbefilledintoaPetri-dish of85mm
diameter,resulting inadepthof2.6mmAgar.
5.Atestpatternofsevermoisture freewellsshallbecutintheagar
tothebottomoftheplate;thepatternshallconsistofonecentre
wellandsixwellsinacirclearoundit.
Diameter ofcentral well:4mm
Diameter ofperipheral wells:6mm
Distance betweencentral andperipheral wells :3mm
6.Thecentralwellshallbefilledwiththestandard antigen.Theperipheral
wells1and4(according toscheme)arefilledwithknownpositive serum,
thewells2,3,5and6arefilledwithtestsera.Thewellsshallbe
filleduntilthemeniscus disappears .
1
&
6φ02
0
5φ @3
Φ
4
7.Thisresultsinthefollowing amountsofreagents :
Antigen : 32microlitres
Control serum:73microlitres
Testsera: 73microlitres
8.Incubation shallbefor72hoursatroomtemperature (20-27°C)ina
closedhumidchamber.
9.Thetestmaybereadat24and48hoursbutafinalresultmaynotbe
obtained before72hours.
a)atestserumispositiveifitformsaspecificprecipitin linewith
theB.L.V.antigenandformsacompletelineofidentitywiththe'
control serum;

-10-
b)atest serum isnegative ifitdoes not form aspecific line with
theB.L.V. antigen and ifitdoes notbend the line ofthe
control serum ;
c)the reaction would beconsidered inconclusive ifit :
i)bends the line ofthe control serum towards the B.L.V. antigen
well without forming avisible precipitin line with the antigen ,
or
ii)ifitcannot beread either asnegative oraspositive .
Ininconclusive reactions the test may bere-run and concentrated
serum may beutilised .
B.Method for Antigen Standardisation
Necessary solutions and materials :
1.AOmlof1.6% agarose in0.05 MTris /HCLbuffer ,pH7.2 with 8.5% NaCl .
2.15mlofabovine leukosis serum ,having antibody only tobovine leukosis
virus glycoproteins ,diluted 1:10in0.05 MTris /HClbuffer -,pH7.2
with 8.5% NaCl .
3.15mlofabovine leukosis serum ,having antibody only tobovine leukosis
virus glycoproteins ,diluted 1:5in0.05 MTris /HClbuffer ,pH7.2
with 8.5% NaCl .
4.Four plastic petri dishes with adiameter of85mm.
5.Apunch with adiameter of4-6mm.
6.Areference antigen .
7.Theantigen which istobestandardised .
8.Awaterbath (56°C ).
Procédure :
Dissolve theagarose (1.6% )intheTris /HClbuffer bycarefully heating
to100°C .Place in56°C water bath forapproximately 1hour .Also ,place
thebovine leukosis serum dilutions in56°C water bath .
Now,mix15mlofthe56°C agarose solution with the15mlbovine leukosis
serum (1:10),quickly shake andpour 15mlinto 2petri dishes each .
Repeat this procedure with thebovine leukosis serum diluted 1:5.
Whentheagarose hashardened ,holes arepunched according tothefollowing
scheme :

-10a
7Wr/d'»shMo,
Sc'km
<'1
I'OOOOQ
\«3-C.-S>Erctrîcft'shUq2,
Seru,yf
Oo"oO
1»BCV
tOrtdishMo3
St^iM :F7c/•VcfiiifWot
Sert***si:S"

-11-
Addition ofantigen :
I. Petri dishes 1+3
Well A=Undiluted reference antigen
Well B=1:2diluted reference antigen
WellC+E =Reference antigen
Well D=Undiluted antigen tobetested
II.Petri dishes 2+4
Well A=Undiluted test antigen
Well 8=1:2diluted test antigen
Well C=1:4diluted test antigen
Well 0=1:8diluted test antigen
Additional Instructions
1. Theexperiment shall becarried out with two serum dilutions (1:5
and 1:10 )inorder toachieve optimal precipitation .
2. Iftheprecipitation diameter istoo small with both dilutions ,then
the serum shall befurther diluted .
3. Iftheprecipitation diameter inboth dilutions istoo large and
faint ,then alower serum dilution shall bechosen .
4. The final concentration oftheagarose shall be0.8% ;that ofthe
sera 57.and 10%respectively .
5. Plot themeasured diameters inthefollowing coordinate system .
Thedilution oftheantigen tobetested with the same diameter as
thereference antigen istheworking dilution .

-11a-
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-12-
Article 2
ThisDirective shallbeimplemented from1stJanuary 1979.
Article 3
ThisDirective isaddressed totheMemberStates.
DoneatBrussels- For
ThetheCounciI,
President