IIP COMMISSION OF THE EUROPEAN COMMUNITIES 
mm 
HI COM(90)492 f inal 
Brussels, 9 November 1990 
Proposal for a 
COUNCIL DIRECTIVE 
amending Directive 64/432/EEC as regards the diagnosis of 
bovine brucellosis and enzootic bovine leukosis 
(presented by the Commission) 
- 2 -
EXPLANATORY MEMORANDUM 
Recent advances in Scientific knowledge have allowed for the development of 
new tests (ELISA) for the diagnosis and control of bovine brucellosis and 
for the adaptation of tests for the detection of enzootic bovine leukosis. 
A scientific group (Brucellosis uniformity antigen group) have studied the 
matter and have carried out a number of international scientific trials, 
resulting from which recommedations have been made to Incorporate the ELISA 
as an alternative for milk tests for the maintenance of officially 
brucellosis free herds as well as individual blood samples. 
The attached is designed to accomplish the abovementioned objectives. 
There are no financial repercussions on the Community budget involved. 
- 3 -
Proposal for a 
COUNCIL DIRECTIVE 
amending Directive 64/432/EEC as regards the diagnosis of 
bovine brucellosis and enzootic bovine leukosis 
THE COUNCIL Of THE EUROPEAN COMMUNITIES, 
Having regard to the Treaty establishing the European Economic Community, 
and in particular Article 43, 
Having regard to the proposal from the Commission, 
Having regard to the opinion of the European Parliament, 
Having regard to the opinion of the Economic and Social Committee, 
Whereas Council Directive 64/432/EEC of 26 June 1964 on animal health 
problems affecting Intra-Communlty trade In bovine animals and swine*1), 
as last amended by Directive 90/422/EEC*2), lays down the methods for 
maintaining the status of officially brucellosis free herds and enzootic 
bovine leukosis free herds; 
Whereas, due to new scientific knowledge and technical developments In the 
diagnosis and control of bovine brucellosis and enzootic bovine leukosis, 
an adjustment of existing Community measures In this field has proved 
necessary; 
(1) OJ No 121, 29.7.1964, p. 1977/64 
(2) OJ No L 224, 18.8.1990, p. 9. 
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HAS ADOPTED THIS DIRECTIVE: 
Article 1 
The Annexes to Directive 64/432/EEC are hereby amended in accordance with 
the Annex to this Directive. 
Article 2 
Member States shall bring into force the laws, regulations and 
administrative provisions necessary to comply with this Directive not later 
than 1 January 1991. They shall forthwith inform the Commission thereof. 
When Member States adopt these provisions, these shall contain a reference 
to this Directive or shall be accompanied by such reference at the time of 
their official publication. The procedure for such reference shall be 
adopted by Member States. 
Article 3 
This Directive is addressed to the Member States. 
Done at Brussels, 
For the CounciI, 
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oHrifcÀ, 
t. Annex A, 11.A.1.(c)(ii) is replaced by the following: 
(il) are checked annually to establish that brucellosis Is not 
present by three ring tests or milk EL ISA carried out at 
intervals of at least three months or two ring tests or milk 
EL ISA at an interval of at least three months and one 
serological test (sero-agglutination test or buffered brucella 
antigen test or plasma agglutination test or plasma ring test 
or micro-agglutination test or individual blood ELISA) carried 
out at not less than six weeks after the second ring test or 
milk ELISA. If ring tests or milk ELISA are not carried out, 
two serological tests (sero-agglutiantIon test or buffered 
brucella antigen test or plasma agglutination test or plasma 
ring test or micro-agglutination test or individual blood 
ELISA) shall be carried out each year at Intervals of at least 
three months and not more than six months. 
Where, in a Member States or region thereof in which all 
bovine herds are subject to official operations to combat 
brucellosis, not more than 1% of bovine herds are Infected, it 
shall be sufficient to carry out each year two ring tests or 
two milk ELISA at an Interval of at least threee months, or 
one serological test (sero-agglutination test or buffered 
brucella antigen test or plasma-agglutination test or plasma-
ring test or micro-agglutination tests or individual blood 
ELISA). 
Where ring tests are carried out on bulk tanks, the number of 
tests referred to In the preceding subparagraphs shall be 
doubled and the intervals between the tests shall be halved." 
2. The following point shall be added to Annex C: 
"H. The ELISA for brucellosis as described under Annex G" 
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3. In Annex G: 
a) The following words shall be added to the title: 
"and brucellosls" 
b) Point C is replaced by the following: 
"C. Enzyme-linked immunosorbent assay (ELISA) for enzootic 
bovine leukosis and bovine brucellosis. 
1. For the ELISA method, the materials and reagents 
to be used are as follows: 
(a) solid phase microplates, cuvettes or any 
other sol id phase; 
(b) the antigen Is fixed to the solid phase with 
or without the aid of polyclonal or 
monoclonal catching antibodies, if antigen 
is coated directly to the solid phase all 
test samples giving positive reactions have 
to be retested against control antigen in 
the case of E.B.L.. The control antigen 
should be identical to the antigen except 
for the B.L.V. antigens. If catching 
antibodies are coated to the solid phase the 
antibodies must not react to other antigens 
other than B.L.V. or brucellosis antigens; 
(c) the biological fluid to be tested; 
(d) a corresponding positive and negative 
control ; 
(e) conjugate; 
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(f) a substrate adapted to the enzyme used; 
(g) a stopping solution, if necessary; 
(h) solutions for the dilution of the test 
samples for preparations of the reagents and 
for washing; 
(1) a reading system appropriate to the 
substrate used. 
2. Standardization and sensitivity of test: 
(a) For enzootic bovine leukosis the sensitivity 
of the EL I SA assay must be of such a level 
that E4 serum is scored positive when 
diluted 10 times (serum samples) or 250 
times (milk samples) more than the dilution 
obtained of individual samples when these 
are included in pools. 
In assays where samples (serum and milk) are 
tested Individually E4 serum diluted 1 to 10 
(In negative serum) or 1 to 250 (in negative 
milk) must be scored positive when tested in 
the same assay dilution as used for the 
individual test samples. 
The E4 serum will be supplied by the 
National Veterinary Laboratory, Copenhagen. 
(b) (i) For brucellosis, bulk milk samples are 
classified negative if they give a 
reaction less than fifty percent of 
that given by a 1 in 10 000 dilution 
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of the second International 
Brucellosis Standard serum made up in 
negatIve milk; 
(II) for brucellosis individual serum 
samples are classified negative if 
they give a reaction less than ten 
percent of that given by a 1 in 200 
dilution of the second International 
Brucellosis Standard serum made up in 
sal I ne so I ut ion. 
The brucellosis ELISA standards shall 
be as specified in Annex C, A 1 and 2 
(to be used at the dilutions indicated 
on the label). 
3. Conditions for use of the ELISA test for E.B.L. 
The ELISA method may be used on a sample of milk 
or whey taken from the ml Ik collected from a farm 
with at least 30% of dairy cows in milk. 
If use is made of one of these abovement ioned 
possibilities, measures must be taken to ensure 
that the samples taken can be identified with the 
animals from which the milk or sera examined were 
taken. 
If one of the samples scores positive, the herd 
must remain under official supervision until a 
negative result has been recorded for at least two 
individual tests carried out, at a minimum 
Interval of four months, on all cattle aged more 
than six months, In accordance with the 
abovementioned provisions and In a laboratory that 
is directly supervised by a laboratory mentioned 
in point A." 
T»*k *orce PME 
XOMMTITIVENgSS AND EMPLOYMENT IMPACT STATEMENT 
ii — • • — y— «•• i——^—« T»*k *orce PME 
XOMMTITIVENgSS AND EMPLOYMENT IMPACT STATEMENT 
II • • — y— «•• i——^—« 
I. what ic the sj'.n reason fur Introducing the measure ? 
Tu use advanced technology to diaonose ch" *•»*** 
II. Features nf i.K« businesses in Question. Ill particular : 
<*> »rt there many SMEs ? 
NO 
•<b) Are Uiey cnnt.irnirated in region* which at >• -. 
i. eligible tor regional airl in the Member atotcs ? 
H. eligible under ihp €*0F ? 
III. Whet direct obligations does this neesure t«pos« on business** 1 
-NONE 
IV. What indirect obligation* are locel authorities LikvLy to Impott , • '!";1 
on businesses ? 
NONb 
n 
V. Arr Tti«r« any special Measures in rrKpeec of SiMEs 7 Please specify. • -
WO •"'"';. 
Vi„ What is th* ;.ik»ly effect on : 
(a) the competitiveness of businesses ? 
NONE 
(b> «mployment 7 
•. none 
VII. Have both sides of industry been consulted 1 
Pl*<*i» indicate ir>e"»r opinions. m0t necessary 

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