,!$. i\ & 
V,ff ,'"1', Itrl(h /*( 
•iÉlSiÉiils COMMISSION OF THE EUROPEAN COMVTUNITIES 
C0M(93) 698 final 
Brussels, 7 January 1994 
Sj^X^ ?-k#&Sf4y 
fr'" «V 
Ci' Proposal for a 
COUNCIL DIRECTIVE 
amending and updating Directive 64/432/EEC 
on health problems affecting intra-Community trade 
in bovine animals and swine 
(presented by the Commission) 
- I -
EXPLANATORY MEMORANDUM 
Recently, Community legislation has been adopted to control Foot and Mouth 
disease, Exotic diseases, and African Swine Fever. It is therefore no longer 
appropriate to retain control provisions for these diseases in Directive 
64/432/EEC which deals with intra-Community trade in live pigs and bovines. 
It is necessary to remove these sections from Directive 64/432/EEC and make 
reference to the relevant control Directive. 
In the light of the Single Market, it is necessary to remove the requirement 
that animals undergo a minimum residency period of six months in a Member 
State prior to each movement. In addition, calves aged under 15 days should 
be brought within the scope of the Directive and references to frontier posts 
should be removed. 
The requirement to carry out premovement tests on animals moving from 
countries or regions recognised as free of Tuberculosis, Brucellosis and 
Enzootic Bovine Leukosis and to carry out mastitis tests on dairy cows should 
also be removed as a further step towards the Single Market. 
The Directive has been amended on over forty occasions since 1964 and it is 
now appropriate that it be updated. The opportunity has also been taken to 
simplify and clarify the provisions of the Directive. 
The attached proposal is designed to accomplish these objectives. 
There are no financial repercussions on the Community budget involved. 
- va -
Proposal 
for a 
CounciI Directive 
of 
amending and updating Directive 64/432/EEC 
on health problems affecting intra-Community trade 
in bovine animals and swine 
THE COUNCIL OF THE EUROPEAN UNION. 
Having regard to the Treaty establishing the European Community, and 
in particular Article 43, 
Having regard to the proposal from the Commission,1 
Having regard to the opinion of the European Parliament,2 
Having regard to the opinion of the Economic and Social Committee,3 
Whereas important progress has been made in the harmonisation of the veteri­
nary field; in particular the Council has adopted Directive 90/425/EEC of 26 
June 1990 concerning veterinary and zootechnical checks applicable in 
intracommunity trade in certain live animals and products with a view to the 
completion of the internal market4, Directive 91/496/EEC of 15 Juiy >991 
laying down the principles governing the organisation of veterinary checks on 
animals entering the Community from third countries5, Directive 85/511/EEC of 
18 November 1985 introducing Community measures for the control of foot and 
mouth disease6 and Directive 92/117/EEC of 17 December 1992 introducing 
general Community measures for the control of certain animal diseases and 
specific measures relating to swine vesicular disease7; 
1 OJ No C 
2 OJ No C 
3 OJ No C 
4 OJ No L 224, 18.08.1990, p.29 
5 OJ No L 268, 24.09.1991, p.56 
6 OJ No L 315, 26.11.1985, p.11 
7 OJ No L 62, 15.03.1993, p.69 
- 2 -
Whereas it is necessary, in the light of this situation, to modify Council 
Directive 64/432/EEC8 of 26 June 1964 on health problems affecting intra-
Community trade in bovine animals and swine, as last amended by Council 
Directive 92/102/EEC9 of 5 December 1992, in particular concerning the period 
of residence in a Member State prior to movement, rules for trade in animals 
aged under 15 days and rules for control of certain diseases; 
Whereas Directive 64/432/EEC has been substantially amended on several 
occasions; that it is thus advisable for the sake of clarity and rationality 
to bring the Directive up-to-date. 
HAS ADOPTED THIS DIRECTIVE 
Article 1 
Directive 64/432/EEC is hereby replaced by the text in Annex I to this 
Direct ive. 
Article 2 
Member States shall bring into force the laws, regulations and administrative 
provisions necessary to comply with this Directive not later than 1 July 
1994. They shall forthwith inform the Commission thereof. 
When Member States adopt these measures, they shall contain a reference to 
this Directive or shall be accompanied by such reference on the occasion of 
their official publication. The methods of making such a reference shall be 
laid down by the Member States. 
Article 3 
This directive is addressed to the Member States 
8 OJ No L 121, 29.07.1964, 1977/64 
9 OJ No L 355, 5.12.1992, p.32 
- 3 -
ANNEX I 
Article 1 
This directive shall apply to intra-community trade in bovine animals and 
swine. 
Article 2 
For the purposes of this directive : 
(a) holding : shall mean a holding as defined in Article 2 of Council 
Directive 90/425/EEC. 
(b) animal for slaughter : 
means a bovine animal (including the species Bubalus bubalus) or 
swine intended to be taken to a slaughterhouse or market from which 
it may only move to slaughter: 
(c) animals for breeding or production : 
means bovine animals (including the species Bubalus bubalus) and 
swine other than those referred to in (b), including those intended 
for breeding, milk or meat production, or draft purposes; 
(d) officially tuberculosis-free bovine holding : 
means a bovine holding which satisfies the conditions laid down in 
Annex A.I 1.2 and 3: 
(e) officially tuberculosis-free Member State or part of a Member State : 
means a Member State or part of a Member State which satisfies the 
conditions laid down in Annex A.I 4.5 and 6: 
(f) officially brucellosis-free bovine holding : 
means a bovine holding which satisfies the conditions laid down in 
Annex A I I 1.2 and 3.; 
- 4 
(g) officially brucellosis-free Region : 
means a part of a Member State which satisfies the conditions laid 
down in Annex A II 7.8 and 9.; 
(h) officially brucellosis-free Member State : 
means a Member State which satisfies the conditions laid down in Annex 
A II 10.11 and 12.; 
(i) brucellosis-free bovine holding : 
means a bovine holding which satisfies the conditions laid down in 
Annex A 11 4.5 and 6; 
(J) Enzootic bovine leucosis free holding : 
means a holding which satisfies the conditions laid down in Annex D. 
Chapter 1. Sections A and B. 
(k) enzootic bovine free Member State or region : 
means a region or Member State which meets the requirements laid down 
in Annex D. Chapter I sections E.F and G. 
(I) official veterinarian : 
means the veterinarian designated by the competent central authority 
of the Member State; 
Art icle 3 
1. Each Member State shall ensure that only animals that fulfil the conditions 
laid down in this Directive, where applicab le. are sent from its territory 
to that of another Member State. 
2. Bovine animals and swine covered by this directive must : 
(a) be subjected to a health examination and identity check by an official 
veterinarian and show no clinical sign of disease; 
- 5 -
(b) not have been obtained from a holding or an area which for health 
reasons is subject to prohibition affecting the species involved in 
accordance with Community or National legislation. 
(c) be identified as provided for in Council Directive 92/102/EEC; 
(d) not be animals which are to be slaughtered under a contagious or 
infectious disease eradication programme of a Member State. 
(e) comply with the provisions of Article 4 and Article 5. 
Art icle 4 
1. Bovine animals and swine covered by this directive must not, at any time 
from leaving the holding of origin to arrival at the destination in another 
Member State, come in contact with cloven hoofed animals other than animals 
that fulfil the conditions for intracommunity trade. 
2. Bovine animals and swine covered by this directive must be transported in 
means of transport which have first been cleansed and disinfected with a 
disinfectant officially authorized in the Member State of Origin. The 
transport vehicles must be so arranged that the animals' faeces, litter or 
fodder cannot flow or fall out of the vehicle during transportât!c:. 
3. Rules for the approval of sites where disinfection may be carried out and 
the procedures necessary to ensure and verify compliance with the 
requirements of paragraph 3 above shall be determined in accordance with 
the provisions of Article 12. 
- 6 -
Art icle 5 
1• Bovine animals and swine covered by this directive must be accompanied 
during transportation to the destination by a health certificate conforming 
to Annex F. The certificate shall consist of a single sheet and shall 
contain a serial number. It shall be drawn up on the day of the health 
examination, in one of the official languages of the country of destination 
at least. The certificate shall be valid for 10 days from the date of the 
health examination. However, where the health examination takes place after 
leaving the holding of origin, as provided for in paragraph 2 below, the 
certificate shall be valid for 10 days after leaving the holding of origin. 
2. The health examination and the issuing of the health certificate for a 
consignment of animals may be carried out in the holding of origin or at 
the point of loading. In the latter case, the certifying official 
veterinarian must have been provided with the evidence issued by the 
official veterinarian for the holding of origin either comprising an 
official document containing the necessary information or in the form of 
the certificate contained in Annex F with Sections A and B duly completed 
and certified. The address of any interim location such as an approved 
market or approved assembly point (part C) must also be completed by the 
official veterinarian responsible for the holding of origin. 
3. The official veterinarian responsible for the interim location shall 
certify arrival of the animals there and where applicable complete the 
final intracommunity certification (Annex F. Part D). Where animals do not 
enter an interim location the final intracommunity certification shall be 
completed by the official veterinarian responsible for the holding of 
origin. 
4- The official veterinarian completing Annex F. part D shall be responsible 
for registering the movement on the AN IMP system. 
5. Interim locations used in intracommunity trade such as markets or assembly 
centres must : 
- 7 -
a. be supervised by an official veterinarian who shall ensure that, in 
particular, the provisions of Article 4.1 and Article 4.2 are complied 
with and who shall provide the necessary certification referred to 
above. The official veterinarian's duties may be further specified by a 
decision taken under the procedure in Article 12 
b. be located in an area which is not subject to prohibition in accordance 
with Community legislation 
c be cleaned before use and disinfected, as required by the official 
veterinarian responsible for the premises. 
d. be designated as approved markets and assembly centres by the Member 
State of origin. Such approval may be limited to a particular species or 
to animals for breeding and production or to animals for slaughter. The 
Member State shall notify the competent central authorities of the other 
Member states and the Commission as to which markets or assembly centres 
are approved. 
6. The interim location may also be an officially approved market located in a 
Member State which is not the Member State of destination, in this case, 
the certificate in Annex F (including section D) must be completed by the 
official veterinarian responsible in the Member State where the animals 
originate. The official veterinarian responsible for the market shall 
provide certification to the animals destination by completing a second 
certificate, as in Annex F. endorsing it with the serial number of the 
original and attaching it to the original or to a copy of the original 
cert ifIcate. 
- 8 -
Article 6 
1. Animals for breeding or production must, in addition to the requirements in 
Art icles 3.4 and 5: 
- have remained in the holding of origin for a period of 30 days prior to 
loading or since birth where the animals are aged less than 30 days. The 
official veterinarian must, on the basis of the official identification 
provided for in Article 3.2c and official records be satisfied that the 
animals have complied with this condition and furthermore that the 
animals have originated in the Community or been imported from a Third 
Country in compliance with Community animal health legislation. 
- Animals imported from a third country into a Member State which is not 
that of ultimate destination, must be transported to the Member State of 
destination as quickly as practicable under cover of certification 
issued under Article 7 of Council Directive 91/496/EEC. Upon arrival at 
the destination, such animals must, as regards any further movements 
submit to the requirements of this directive and in particular to the 
residency requirement in the first indent. 
2. Bovine animals for breeding and production must, in addition to the 
requirements in Articles 3. 4 and 5: 
(a) come from an officially tuberculosis-free bovine holding and in the 
case of animals more than six weeks old, have reacted negatively to an 
intradermal tuberculin test carried out during the 30 days prior to 
leaving the herd of origin, in accordance with the provisions of Annex 
B point 32.d: 
This intradermal tuberculin test is not required if the animals 
originate in a Member State or part of a Member State recognised as 
Officially Tuberculosis Free. 
- 9 -
(b) in the case of non castrated animais come from an officially 
brucellosis-free bovine holding and if more than 12 months old, have 
shown a brucella count lower than 30 international units of 
agglutination per millilitre when given a sero-aggiutination test 
carried out during the 30 days prior to leaving the herd of origin and 
complying with the provisions of Annex C paragraph A; 
This seroagglutination test is not required if the animais originate 
in a Member State or part of a Member State recognised as Officially 
Brucellosis Free. 
(c) come from an enzootic bovine leucosis free holding within the meaning 
of Article 2 (g); 
(d) not, at any time from leaving the holding of origin to arrival at the 
destination, come in contact with animals which only meet the 
requirements in Article 6.3 below. 
3. Bovine animals for slaughter must, in addition to the requirements in 
Articles 3.4 and 5 come from herds that are officially tuberculosis free, 
enzootic bovine leucosis free and in the case of uncastrated bovines, from 
herds that are officially brucellosis free. 
Art icle 7 
Animals for slaughter which have been taken on arrival in the country of 
destination to a slaughterhouse must be slaughtered there as soon as possible, 
in accordance with animal health requirements. 
Animals for slaughter which have been taken on arrival in the Member State of 
destination to a market, under whose rules all animals must be removed after 
the market to a slaughterhouse approved for this purpose by the competent 
central authority, must be slaughtered at that slaughterhouse not later than 
five days after arriving at the market. 
- 10 -
Art icle 8 
Any person or persons suspecting the presence of one of the diseases listed in 
Annex Ed) shall notify the competent authority and, where appropriate, inform 
the owner or keeper of the animals as soon as possible. 
Each Member State shall forward to the Commission by 31 May each year details 
of the occurrence of tuberculosis, brucellosis and enzootic bovine leucosis in 
its territory in the previous calender year and details of the numbers of 
herds in each status category for each disease along with information on the 
monitoring or eradication programmes in operation in each region. The content 
and format of the information required shall be determined by the Commission. 
The Commission shall present this information to the Member States in the 
framework of the Standing Veterinary Committee and in particular may utilise 
it in relation to the decisions mentioned in Annex A and Annex D. 
Art icle 9 
1. A Member State which proposes a compulsory national control programme for 
one of the contagious diseases I isted in Annex EMI) for all or part of its 
territory may submit the said programme to the Commission, outlining in 
part icular: 
- the distribution of the disease in the Member State, 
- the reasons for the programme, taking into consideration the importance 
of the disease and the programme's likely benefit in relation to its 
cost, 
- the geographical area in which the programme will be implemented, 
- the status categories to be applied to the animal establishments, the 
standards which must be attained in each category, and the test 
procedures to be used, 
- 11 -
- the programme monitoring procedures, the results of which must be 
supplied at least annually to the Commission. 
- the action to be taken if, for any reason, an establishment loses its 
status, 
- the measures to be taken if the . esults of the tests carried out in 
accordance with the provisions of the programme are positive. 
2. The Commission shall examine the programmes presented by the Member States. 
Programmes as referred to in paragraph 1 may be approved in compliance with 
the criteria laid down in paragraph 1 in accordance with the procedure 
provided for in Article 12. According to the same procedure, the additional 
guarantees, general or limited, which may be required in intra-Community 
trade, shall be defined at the same time or at the latest three months 
after approval of the programmes. Such guarantees must not exceed those 
which the Member State implements nationally. 
3. Programmes submitted by Member States may be amended or supplemented in 
accordance with the procedure laid down in Article 12. Amendments or 
additions to programmes which have already been approved or to guarantees 
which have been defined in accordance with paragraph 2 may be approved 
under the same procedure. 
Article 10 
1. Where a Member State considers that its territory or part of its territory 
is free from one of the diseases I isted in Annex EMI), it shall present to 
the Commission appropriate supporting documentation, setting out in 
particular: 
- the nature of the disease and the history of its occurrence in its 
territory, 
- 12 -
- the results of surveillance testing based on serological, 
microbiological, pathological or epidemiological investigation and on 
the fact that the disease must by law be notified to the competent 
authorities, 
- the period over which the surveillance was carried out, 
- where applicable, the period during which vaccination against the 
disease has been prohibited and the geographical area concerned by the 
prohibition, 
- the arrangements for verifying the absence of the disease. 
2. The Commission shall examine documentation submitted by Member States. The 
additional guarantees, general or specific, which may be required in intra-
Community trade may be defined in accordance with the procedure laid down 
in Article 12. Such guarantees must not exceed those which the Member State 
implements nationally. 
3. The Member State concerned shall notify the Commission of any change in the 
details specified in paragraph 1 which relate to the disease, in particular 
regarding any new outbreaks of the disease. The guarantees defined as laid 
down in paragraph 2 may, in the light of such notification, be amended or 
withdrawn in accordance with the procedure laid down in Article 12. 
Article 11 
The Annexes to this Directive may be modified in accordance with the procedure 
detailed in Article 12 
In derogation to the provisions of this Directive, specific conditions for 
trade in animals for special purposes, and in particular for show purposes, 
may be determined in accordance with the procedure in Article 12. 
- 13 
Article 12 
1. Where the procedure laid down in this Article is to be used, matters shall 
without delay be referred by the chairman, either on his own initiative or 
at the request of a Member State, to the standing veterinary committee 
(hereinafter called the "committee ") set up by the Council Decision of 15 
October 1968. 
2. Within the committee the votes of Member States shall be weighted as 
provided in Article 148(2) of the treaty. The chairman shall not vote. 
3. The representative of the Commission shall submit a draft of the measures 
to be adopted. The committee shall deliver its opinion on such measures 
within a time limit set by the chairman according to the urgency of the 
matters concerned. Opinions shall be delivered by a majority of 54 votes. 
4. The Commission shall adopt the measures and shall apply them immediately 
where they are in accordance with the opinion of the committee. Where they 
are not in accordance with the opinion of the committee or if no opinion is 
delivered, the Commission shall without delay propose to the Council the 
measures to be adopted. 
The Council shall adopt the measures by a qualified majority. 
If, within three months from the date on which the proposal was submitted to 
it, the Council has not adopted any measures, the Commission shall adopt the 
proposed measures and apply them immediately. 
Article 13 
1. Where the procedure laid down in this Article is to be used, matters shall 
without delay be referred by the chairman, either on his own initiative or 
at the request of a Member State, to the standing veterinary committee 
(hereinafter called the " committee ") set up by the Council decision of 
15 October 1968. 
- 14 -
2. Within the committee the votes of the Member States shall be weighted as 
provided in Article 148 (2) of the treaty. The chairman shall not vote. 
3. The representative of the Commission shall submit a draft of the measures 
to be adopted. The committee shall deliver its opinion on such measures 
within two days. Opinions shall be delivered by a majority of 54 votes. 
4. The Commission shall adopt the measures and shall apply them immediately 
where they are in accordance with the opinion of the committee. Where they 
are not in accordance with the opinion of the committee or if no opinion is 
delivered, the Commission shall without delay propose to the Council the 
measures to be adopted. 
The Council shall adopt the measures by a qualified majority. If within 15 
days from the date on which the proposal was submitted to it, the Council has 
not adopted any measures, the Commission shall adopt the proposed measures and 
apply them immediately. 
Article 14 
The rules laid down in Council Directive 90/425/EEC of 26 June 1990 concerning 
veterinary and zootechnical checks in intra-Community trade in certain live 
animals and products with a view to the completion of the internal market, as 
last amended by Council Directive 92/60/EEC of 30 June 199210 shall apply in 
particular to checks at origin, to the organization of, and follow-up to, the 
checks to be carried out by the country of destination, and to the safeguard 
measures to be implemented. 
Article 15 
This directive is addressed to the Member States. 
10 OJ No L 268 24.09.92 p 75 
ANNEX A 
I. OFFICIALLY TUBERCULOSIS-FREE BOVINE HOLDING 
1. A bovine holding is officially tuberculosis-free if : 
(a) all the animals are free from cli.iical signs of tuberculosis; 
(b) all the animals over six weeks old have reacted negatively to at least 
two official intradermal tuberculin tests carried out in accordance 
with Annex B. the first six months after the elimination of any 
infection from the holding and the second six months later or 
in the case where the herd has been assembled solely from animals that 
originate in Officially Tuberculosis Free herds the first test shall 
be carried out at least 60 days after assembly and the second shall 
not be required 
(c) following the completion of the first test mentioned in (b) above, no 
bovine animal aged over six weeks has been introduced into the holding 
unless it has reacted negatively to an intradermal tuberculin test 
performed and assessed according to Annex B and carried out either in 
the 30 days prior to. or the 30 days after the date of it's 
introduction into the holding. 
This test shall not be necessary in Member States where the percentage 
of bovine holdings infected with tuberculosis is less than 0.2% and 
where the animal originates in an Officially Tuberculosis Free 
holding. 
2. A bovine holding will retain Officially Tuberculosis Free status if; 
(a) the conditions detailed in 1.(a) and 1.(c) continue to apply 
- <6 
(b) all animals entering the holding come from holdings of the Officially 
Tuberculosis Free status. 
(c) all animals in the holding, with the exception of calves aged under 
six weeks which were born in the holding, are subjected to routine 
tuberculin testing in accordance with Annex B at yearly intervals. 
However, the Commission, in accordance with the procedure in Article 
12. may, for a Member State or part of a Member State where all the 
bovine holdings are subject to official operations to combat 
tuberculosis, alter the frequency of the routine tests as follows; 
if the percentage of bovine holdings infected with tuberculosis is not 
more than 1% on average during the two most recent annual supervisory 
periods, the interval between routine holding tests may be increased 
to two years; 
if the percentage of infected bovine holdings is not more than 0.2% on 
average during the two most recent biennial supervisory periods, the 
interval between routine tests may be increased to three years; 
if the percentage of infected bovine holdings is not more than 0.1% on 
average during the two most recent triennial supervisory periods, the 
interval between routine tests may be increased to four years and/or 
the age at which animals have to undergo these tests may be increased 
to 24 months. 
The Commission may also in accordance with Article 13. take a decision 
increasing the freguency of routine tuberculin testing if the level of 
disease appears to have increased. 
3. The Officially Tuberculosis free status of a holding shall be suspended if; 
(a) the conditions detailed in 2 above have not been complied with. 
(b) an animal is deemed to have reacted positively to a routine tuberculin 
test, or a case of tuberculosis has been diagnosed at routine post 
mortem examination. 
- l-( 
In these cases, the status shall remain suspended until such time as all 
the remaining animals over six weeks of age have reacted negatively to 
at least two official intradermal tuberculin tests in accordance with 
Annex B. the first one carried out at least two months after elimination 
of the animal from the holding and the second one at least 42 days after 
the first. 
However where the routine holding test detailed in 2(c) above has not 
been performed on time, the status of the holding shall not be suspended 
provided that the test is carried out not later than 60 days after it 
was originally due, and provided that subsequent testing takes place 
according to the original timetable. 
(c) the holding contains animals of unresolved status as described in 
Annex B point 32. In this case the status of the holding shall remain 
suspended until the animals status has been clarified. 
4- A Member State or part of a Member State may be declared Officially 
Tuberculosis Free according to the procedure in Article 12 if it meets the 
following conditions; 
a. the percentage of infected bovine holdings has not been more than 0.01% 
for 6 consecutive years and at least 99.9% of the holdings have been 
declared Officially Tuberculosis Free for 10 years 
b. an identification system making it possible to identify the herds of 
origin and transit for each bovine animal is in existence 
c. all bovine animals slaughtered must be subjected to a post mortem 
examination by an official veterinarian. 
v ai. suspected cases of tuberculosis must La fully investigated, 
including tracing and checking any herds of origin or transit and 
carrying out all appropriate laboratory examinations. While such 
examinations take place the officially tuberculosis free status of the 
herds of origin or transit shall be suspended until clinical or 
laboratory examinations or tuberculin tests have ruled out the presence 
of bovine tuberculosis. 
^ 
5. The Member State or part of a Member State will retain Officially 
Tuberculosis Free status If: 
a. The conditions 4.a to 4.d above continue to apply 
b. when a case of tuberculosis is confirmed, the officially tuberculosis 
free status of the holding of orioi.i and transit is withdrawn. 
c. the Officially Tuberculosis free status of holdings where tuberculosis 
has been confirmed remains withdrawn until; 
- all the animals that have been deemed to be infected have been 
slaughtered 
- disinfection of the premises and utensils has taken place 
- all the remaining bovine animals over six weeks of age have reacted 
negatively to at least two official intradermal tests in accordance 
with Annex B. the first at least six months after the removal of the 
Infected animals, the second six months after the first. 
6. If there is evidence of a significant change in the situation as regards 
tuberculosis in a Member State or part of a Member State which has been 
recognised as Officially Tuberculosis Free, the Commission may in 
accordance with Article 13 take a decision suspending or revoking the 
status and requiring routine tuberculin tests to be carried out in 
accordance with one of the schedules in 2.d above. 
' *\ 
I I. OFFICIALLY BRUCELLOSIS FREE AND BRUCELLOSIS FREE BOVINE HOLDINGS 
For the purposes of this Annex A.11 "bovine animals" means all bovine animals 
with the exception of males castrated before the age of four months. 
1. A bovine holding Is Officially Brucellosis Free if; 
(a) it contains no bovine animals which have been vaccinated against 
brucellosis, except females which have been vaccinated at least three 
years previously; 
(b) all the bovine animals have been free from clinical signs of 
brucellosis for at least six months; 
(c) all the bovine animals over 12 months old have been subjected to one 
of the following test regimes with negative results in accordance with 
Annex C; 
( •) two seroagglutinat ion tests at an interval of more than three 
months and less than twelve months 
(ii) three ring tests at three monthly intervals followed at least 
six weeks later by a seroagglutination test 
(iii) two buffered brucella antigen tests at an interval of more 
than three months and less than twelve months 
( iv) two micro-agglutination tests at an interval of more than 
three months and less than twelve months. 
2. A bovine holding will retain Officially Brucellosis Free status if; 
(a) one of the following test regimes is carried out annually with 
negative result in accordance with Annex C; 
( i ) three ring tests carried out at intervals of at least three 
months 
- cJtt-
( i i) three milk ELISA's carried out at intervals of at least three 
months 
(iii) two ring tests carried out at an interval of at least three 
months followed at least six weeks later by a serological test 
(iv) two milk ELISA's carried out at an interval of at least three 
months followed at least six weeks later by a serological test 
(v) two serological tests carried out at an interval of at least 
three months and not more than six months. 
However, the Commission, in accordance with the procedure in Article 
12. may, for a Member State or part of a Member State which is not 
Officially Brucellosis Free but where all the bovine holdings are 
subject to official operations to combat brucellosis, alter the 
frequency of the routine tests as follows; 
- where not more than 1% of bovine holdings are infected, it may be 
sufficient to carry out each year two ring tests or two milk Elisa 
at an interval of at least three months, or one serological test. 
- where at least 99.8 % of bovine holdings have been recognized as 
Officially Brucellosis Free for at least four years, the interval 
between checks may be extended to two years and the checks must be 
carried out using one of the serological tests referred to in 
paragraph 7.(a) below. 
(b) all bovine animals entering the holding come from holdings of 
Officially Brucellosis Free status, and in the case of bovine animals 
over 12 months old, have shown a brucella count of less than 30 iu of 
agglutination per ml when given a sero-agglutination test in 
accordance with Annex C during the 30 days prior to introduction into 
the holding: 
However the sero-agglutination test described above need not be 
required in Member States, or Regions of Member States, where the 
percentage of bovine holdings infected with brucellosis has not 
exceeded 0.2 % for at least two years and where the animal comes from 
an official brucellosis-free bovine holding within that Member State 
or Region and has not during transportai ion come into contact with 
bovine animals of lesser status. 
(c) Notwithstanding 2b above, bovine animals from a Brucellosis Free 
bovine holding may be introduced into an Officially Brucellosis Free 
holding if they are at least 18 months old and if vaccinated against 
brucellosis, the vaccination was carried out more than a year 
previously. 
Such animals must have shown in the 30 days prior to introduction, a 
brucella count lower than 30 iu of agglutination per ml and a negative 
result when given a complement fixation test, both in accordance with 
Annex C. 
If. however, a bovine animal from a Brucellosis Free holding is 
introduced into an Officially Brucellosis Free bovine holding, under 
these provisions, that holding shall be considered to be Brucellosis 
Free for two years from the date on which the animal was introduced. 
3. The Officially Brucellosis free status of a holding may be suspended or 
withdrawn if: 
(a) the conditions detailed in paragraphs 1 and 2 above have not been 
complied with or 
(b) as a result of laboratory tests or on clinical grounds one or more 
bovine animals are suspected of having brucellosis 
If one or more bovine animals in an Officially Brucellosis Free holding are 
suspected of having brucellosis, the status of the holding may be 
suspended, rather than withdrawn, if the animal or animals are immediately 
destroyed or isolated. 
Jt'ï 
Where the animal has been destroyed, the suspension may be lifted if two 
sero-agglutination tests, carried out in accordance with Annex C on all 
bovine animals in the holding over 12 months old, show a count lower than 
30 iu of agglutination per ml. The first test shall be carried out at least 
30 days after the removal of the animal and the second at least 60 days 
later. 
Where the animal has been isolated, it may be reintroduced into the holding 
and the status of the holding may be restored, if it subsequently shows a 
sero-agglut inat ion count lower than 30 iu of agglutination per ml and has 
given a negative result to a complement fixation test, these tests being 
carried out in accordance with Annex C. 
Where, as a result of laboratory tests or epidemiological investigations, 
brucella infection has been confirmed in a holding, the status of that 
holding shall not be restored until all bovine animals that were pregnant 
at the time of the outbreak have given negative results to the above tests, 
the final test having being carried out at least 21 days after calving. 
4. A bovine holding is Brucellosis Free if it complies with the conditions in 
1a. 1b. and 1c. except that: 
(i) female bovine animals may be vaccinated: 
- before the age of six months old with live strain 19 vaccine or 
other vaccines approved under the procedure laid down in Article 12 
Pi, 
- before the age of 15 months old with killed 45/20 adjuvant vaccine 
which has been officially inspected and recognised: 
(i i) bovine animals aged under 30 months which have been vaccinated with 
live strain 19 vaccine may give a serum agglutination test result 
greater than 30 i.u. but less than 80 i.u. of agglutination per 
millilitre provided that, on the complement fixation test, they give a 
result less than 30 EEC units in the case of females vaccinated less 
than 12 months previously or less than 20 EEC units in all other 
cases. 
l^ 
(iii) in addition to those tests listed in 1(c). the following test 
regimes will also be approved in order to attain Brucellosis Free 
status; 
a. two buffered brucella antigen tests carried out at an interval of 
more than three months and less than 12 months 
b. two micro-agglutination tests carried out at an interval of more 
than three months and less than 12 months 
in accordance with the provisions of Annex C. 
5. A bovine holding will retain Brucellosis Free status if: 
(i) it is subject to one of the testing regimes listed in 2(a) above 
(i i) bovine animals entering the holding comply with the reouirements of 
2(b) above or 
- come from holdings of Brucellosis Free status, and in the case of 
bovine animals over 12 months old, have shown, in the 30 days prior 
to introduction Into the holding, less than 30 iu of agglutination 
per ml when given a sero-agglut inat ion test and a negative 
complement fixation test in accordance with Annex C or. 
- come from holdings of Brucellosis Free status, be aged under 30 
months and have been vaccinated with live strain 19 vaccine may give 
a serum agglutination test result greater than 30 i.u. but less than 
80 i.u. of agglutination per millilitre provided that, on the 
complement fixation test, they give a result less than 30 EEC units 
in the case of females vaccinated les s than 12 months previously or 
less than 20 EEC units in ail other cases. 
— o^Ly 
6. The Brucellosis free status of a holding shall be suspended or withdrawn 
il: 
(a) the conditions detailed in paragraph 4 and paragraph 5 above have not 
been complied with or 
(b) as a result of laboratory tests or on clinical grounds one or more 
bovine animals aged over 30 months are suspected of having brucellosis 
If one or more bovine animals, aged over 30 months, in an Brucellosis Free 
holding are suspected of having brucellosis, the status of the holding may 
be suspended, rather than withdrawn, if the animal or animais are 
immediately destroyed or isolated. 
Where the animal has been destroyed, the suspension may be lifted if two 
sero-agglutination tests, carried out in accordance with Annex C on all 
bovine animals in the holding over 12 months old, show a count lower than 
30 iu of agglutination per ml. The first test shall be carried out at least 
30 days after the removal of the animal and the second at least 60 days 
later. 
Where the animal has been isolated, it may be reintroduced into the holding 
and the status of the holding may be restored, if it subseguently shows a 
sero-agglut inat ion count lower than 30 iu of agglutination per ml and has 
given a negative result to a complement fixation test, these tests being 
carried out in accordance with Annex C. 
Where, as a result of laboratory tests or epidemiological Investigations, 
brucella infection has been confirmed in a holding, the status of that 
holding shall not be restored until all bovine animals that were pregnant 
at the time of the outbreak have given negative results to the above tests, 
the final test having being carried out at least 21 days after calving. 
-2 s 
7. A Region of a Member State may be declared Officially Brucellosis Free 
according to the procedure In Article 12 If it meets the following 
conditions: 
a. no case of abortion due to brucella infection has been recorded for at 
least three years and at least 99.8% of the holdings have been declared 
Officially Brucellosis Free for 10 /ears 
b. an identification system making it possible to identify the herds of 
origin and transit for each bovine animal is in existence 
8. Subject to point 9 below, a region declared Officially Brucellosis Free 
shall retain this status if all bovine animals over 24 months old are 
subjected to either two ring tests or one serological test every three 
years. In the event of a positive result the provisions of point 6 above 
shall apply. 
9. A region declared Officially Brucellosis Free shall report the occurrence 
of all cases of Brucellosis to the Commission. The Commission may according 
to the procedure in Article 13 propose that the status be suspended or 
revoked and recuire that routine brucellosis testing be carried out in 
accordance with one of the schedules in paragraph 2 above. 
10. A Member State may be declared Officially Brucellosis Free according to 
the procedure In Article 12 if it meets the following conditions; 
a. no case of abortion due to brucella infection has been recorded for 
at least three years and at least 99.8% of the holdings have been 
declared Officially Brucellosis Free for 10 years 
b. an identification system making it possible to identify the herds of 
origin and transit for each bovine animal is in existence 
M 
11. A Member State declared Officially Brucellosis Free shall retain this 
Status if; 
- every bovine animal suspected of being infected with brucellosis is 
notified to the competent authority and undergoes official 
investigation for brucellosis including at least two serological blood 
tests including the complemei.c fixation test as well as a 
microbiological examination of appropriate samples taken in the case 
of an abort ion 
- during the period of suspicion which shall continue until negative 
results have been obtained from the tests provided for in the first 
indent, the officially Brucellosis Free status of the herd of origin 
or transit of the suspected bovine shall be suspended. 
- in the event of a positive result, the provisions of point 6 above 
apply. 
12. A Member State declared Officially Brucellosis Free shall report the 
occurrence of all cases of Brucellosis to the Commission. The Commission 
may, according to the procedure in Article 13 propose that the status be 
suspended or revoked and require that routine brucellosis testing be 
carried out in accordance with one of the schedules in paragraph 2 
above. 
13.(a) For the purposes of this Annex A.M. a serological test shall mean 
either a serum agglutination test, buffered brucella antigen test, 
complement fixation test, plasma agglutination test, plasma ring test, 
micro agglutination test or individual blood ELISA. as described in 
Annex C. 
(b) Where ring tests are carried out on bulk tanks, the number of those 
tests referred to in this Annex shall be doubled and the intervals 
between the tests shall be halved. 
ANNEX B 
(Standards for the manufacture and use of bovine and avian tuberculins) 
1. Officially supervised tuberculin tests must be carried out with PPD or HCSM 
tuberculins. 
2. Manufacturers' working standards for the control of bovine PPD and HCSM 
tuberculins must be calibrated in community tuberculin units (CTU) 
following biological assay against the appropriate EEC standard tuberculin. 
3. Manufacturers' working standards for the control of avian tuberculins must 
be calibrated in international units following biological assay against the 
EEC standard for PPD of avian tuberculin. 
4. The EEC standard for PPD of bovine tuberculin is that supplied by the 
Centraal Diergeneeskundig Instituut, Afdeling Rotterdam, The Netherlands. 
5. The EEC standard for bovine HCSM tuberculin is that supplied by the 
Institut Pasteur, Paris, France. 
6. The EEC standard for avian tuberculin is that supplied by the Central 
Veterinary Laboratory, Weybridge, Surrey, England. 
7. Bovine tuberculins must be prepared with one of the mycobacter ium bovis 
strains indicated below : 
(a) AN5; 
(b) Vallée. 
8. Avian tuberculins must be prepared with one of the mycobacter ium avium 
strains indicated below : 
(a) D4ER; 
(b) TB56. 
'« 
9. The ph of tuberculins must be between 6,5 and 7,5. 
10. Antimicrobial preservatives or other substances that may be added to a 
tuberculin shall have been shown, to the satisfaction of the state 
institute responsible for the official testing of the tuberculin, not to 
impair the safety and effectiveness of the product. 
The following are the maximum permitted concentrations for phenol and 
glycerol : 
(a) phenol : 0,5 % m/v; 
(b) glycerol : 10 % v/v. 
11. Provided the tuberculins are stored at a temperature between 2 and 8'C, 
protected from light, they may be used up to the end of the following 
periods subsequent to the last satisfactory potency test : 
(a) liquid PPD tuberculins : two years, 
lyophilized PPD tuberculins :eight years-, 
(b) HCSM tuberculins diluted : two years. 
12. The state institutes listed below shall be responsible for the official 
testing of tuberculins in their respective countries : 
(a) Germany : Paul-Ehrlich institut, Frankfurt/Main; 
(b) Belgium : Instituut voor hygiene enepidemiologie, J.Wytsmanstraat 
14, B-1050 Brussels; 
(c) France Laboratoire national des medicaments vétérinaires, 
Fougères; 
(d) Grand Duchy of Luxembourg : Institute of the supplying country; 
(e) Italy: Istituto superiore di sanita, Rome; 
-=^V 
(f) Netherlands Centraal diergeneeskundig instituut, afde ling 
Rotterdam; 
(g) Denmark Statens Veterinaere Serum laboratorium, Copenhagen 
V; 
(h) Ireland institute of *he supplying country; 
(i) United Kingdom: Central Veterinary Laboratory, Weybridge, Surrey. 
(J) Greece : 
(k) Spain : Laboratorio de Sanidad y produce ion Animal de Granada 
(I) Portugal : Laboratorio Nacional de Investigacao Veeterinaria -
L i sboa 
13. Official testing must be carried out on each batch of bottled 
tuberculins ready for use. 
14. Tuberculins shall be tested by biological and chemical methods. 
15. Tuberculins must be sterile. Tests for sterility shall be carried out 
according to the specifications of the european pharmacopoeia. 
16. A test for the absence of toxic or irritant properties shall be carried 
out according to the specifications of the european pharmacopoeia. 
17. Tuberculins must be chemically analyzed to determine the concentration 
of glycerol and/or phenol and also the concentration of any other 
preservative which may have been added. 
18. A test of non-sensitization to tuberculin must be carried out according 
to the specifications of the european pharmacopoeia. 
V 
19. The potency of tuberculins must be assessed by biological methods. These 
methods must be used for HCSM and PPD tuberculins-, they are based on the 
comparison with standard tuberculins of the tuberculins to be tested. 
20. The protein content of PPD tuberculin must be estimated by the k je IdahI 
method. The nitrogen is converted into tuberculo-protein content by 
multiplying by a factor of 6,25. 
21. The EEC standard for bovine HCSM has a potency of 65 000 community 
tuberculin units (CTU) per ml and is dispensed in ampoules containing 5 
ml of tuberculin. 
22. The EEC standard for bovine PPD has a potency of 50 000 community 
tuberculin units (CTU) per mg of PPD and is dispensed lyoph i I i zed in 
ampoules containing 1,8 mg of PPD, i.e. 0,00002 mg PPD has a potency 
equal to one community tuberculin unit. 
23. The EEC standard for avian PPD has a potency of 50 000 international 
units (i.u.) per mg of the dried material of the purified protein 
derivative and is dispensed in the lyophilized in ampoules containing 
10mg of PPD plus 26,3 mg of salts, i.e. 0,0000726 mg of the standard has 
a potency equal to one international unit. 
24. Tuberculins submitted by manufacturers for testing by the state 
institutes listed in paragraph 12 must have been tested for potency by 
biological assay against the appropriate standards as listed in 
paragraphs 2 and 3. 
25.(a) potency testing on guinea pig 
Albino guinea-pigs weighing between 400 and 600 g must be used. These 
guinea-pigs must be in good health at the time of injection of the 
tuberculin. Not less than eight guinea-pigs shall be used for each 
assay. The assay should be made not less than one month after 
sens it izat ion. 
•V\ 
(aa) for the assay of bovine tuberculins, guinea-pigs shall be sensitised 
by one of the following methods : 
1. The injection of heat-killed mycobacterium bovis strain an5 in 
oiI adjuvant, 
2. The injection of living •Mycobacterium bovis strain an5 in 
physiological saline, 
3. The injection of beg vaccine. 
(bb) for the assay of avian tuberculins guinea-pigs shall be sensitized by 
injection of 2 mg of heat-killed avian-type tubercular bacilli 
suspended in 0,5 ml of sterile liquid paraffin or by the injection of 
live avian-type tubercular bacilli in physiological saline. The avian-
type strain d4 must be used for this purpose. 
(cc) each tuberculin under test shall be assayed against the appropriate 
standard tuberculin by an intradermal assay using groups of guinea-
pigs suitably sensitized. 
The hair shall be clipped from both sides of each guinea-pig. The 
assay shall be carried out by comparing the reactions induced by a 
series of intracutaneous injections of doses of not more than 0,2 ml 
of dilutions of the standard tuberculin in isotonic buffered saline 
solution containing Tween 80, 0,0005%, with a corresponding series of 
injections of the tuberculin under test. Dilutions shall be arranged 
in geometric series, and injected into guinea-pigs according to a 
randomized latin square design (four sites on each side of an eight-
point assay is used). The diameters of the reactions at each site 
should be measured and recorded after 24 to 28 hours. 
For each sample of tuberculin under test, an estimate of relative 
potency against the appropriate standard and its fiducial limits shall 
be made by statistical methods, using the diameters of the reactions 
and the logarithms of the doses as metameters. The bovine tuberculin 
under test is of acceptable potency if its estimated potency 
guarantees per bovine dose 2000 community tuberculin units (more or 
less 25 %) in cattle. The potency of each tuberculin under test shall 
be expressed as appropriate in community tuberculin units or 
international units per ml. 
(b) potency testing on cattle 
Periodic potency testing of bovine tuberculins may be carried out on 
naturally or artificially infected tuberculous cattle. These potency 
tests, on groups of tuberculous cattle, shall be carried out by 
intradermal four or six-point assay of the tuberculin under test 
against the appropriate standard and the potency of the tuberculin 
shall be estimated by statistical methods as in the guinea-pig assay. 
26. The following requirements shall apply to the labelling of tuberculin 
containers and packages : 
The label on the containers and the label on the package shall state : 
- the name of the preparation, 
- for liquid preparations, the total volume in the container, 
- the number of community or international units per ml or per mg, 
- the manufacturer's name, 
- the batch number, 
- the nature and quantity of the reconstituting liquid for the freeze-
drled préparât ion. 
The label on the container or the label on the package shall state: 
- the expiry date, 
- the conditions of storage, 
- the name and, if possible, the proportions of any added substance, 
- the strain of bacillus from which the tuberculin has been made. 
27. Community laboratories designated in accordance with the procedure in 
Article 12 will be made responsible for the additional examination of 
routine issue field tuberculins used in the Member States to ensure that 
the potency of each of these tuberculins is adequate in relation to the 
appropriate community standard tuberculin. These examinations must be 
carried out, in tuberculous bovines, in suitably sensitized guinea-pig^ 
and by appropriate chemical tests. 
28. The following shall be recognized as official intradermal tuberculin 
tests: 
(a) the single intradermal test - this test requires a single injection 
of bovine tuberculin. 
(b) the intradermal comparative tesc - this test requires one injection 
of bovine tuberculin and one injection of avian tuberculin given 
simultaneously. 
29. The dose of tuberculin injected shall be : 
1. Not less than 2 000 CTU of bovine tuberculin; 
2. Not less than 2 000 iu of avian tuberculin.WI5 
The volume of each injection dose shall not exceed 0,2 ml. 
30. Tuberculin tests shall be carried out by injecting tuberculin(s) into 
the skin of the neck. The injection sites shall be situated at the 
border of the anterior and middle thirds of the neck. When both avian 
and bovine tuberculins are injected in the same animal, the site for 
injection of avian tuberculins shall be about 10 cm from the crest of 
the neck and the site for the injection of bovine tuberculin about 12,5 
cm lower on a line roughly parallel with the line of the shoulder or on 
different sides of the neck; in young animals in which there is not room 
to separate the sites sufficiently on one side of the neck, one 
injection shall be made on each side of the neck at identical sites in 
the centre of the middle third of the neck. 
31. The technique of tuberculin testing and interpretation of reactions 
shalI be as fol lows : 
(a) technique : 
Injection sites shall be clipped and cleansed. A fold of skin within 
each clipped area shall be taken between the forefinger and thumb 
and measured with calipers and recorded. A short sterile needle, 
bevel edge outwards, with graduated syringe charged with tuberculin 
attached shall be inserted obliquely into the deeper layers of the 
skin. The dose of tuberculin shall then be injected. A correct 
injection shall be confirmed by palpating a small peal ike swelling 
at each site of injection. The skin-fold thickness of each injection 
site shall be remeasured 72 hours after injection and recorded. 
(b) interpretation of reactions : 
The interpretation of reactions shall be based on clinical 
observations and the recorded increases(s) in skin-fold thickness at 
the sites of injection 72 hours after injection of tuberculin(s). 
(ba) negative reaction : if only limited swelling is observed, with an 
increase of not more than 2 mm in the thickness of the fold of 
skin without clinical signs such as diffuse or extensive oedema, 
exudation, necrosis, pain or inflammation of the lymphatic ducts 
in that region or of the lymph nodes. 
(bb) inconclusive reaction : if no clinical signs such as mentioned in 
(ba) are observed and if the increase in skin-fold thickness is 
more than 2 mm and less than 4mm. 
(be) positive reaction : if clinical signs such as mentioned in (ba) 
are observed or there is an increase of 4 mm or more in the 
thickness of the fold of skin at the injection site. 
32. The interpretation of official intradermal tuberculin tests shall be as 
fo11ows : 
(a) single intradermal test : 
positive : a positive bovine reaction as defined in paragraph 31 
(be); 
inconclusive : an inconclusive reaction as defined in paragraph 31 
(bb); 
negative : a negative bovine reaction as defined in paragraph 
31(ba). 
Animals inconclusive to the single intradermal test shall be subjected 
to another test after a minimum of 42 days. 
Animals which are not negative to this second test shall be deemed to 
be positive to the test. 
Animals positive to the single intradermal test may be subjected to an 
intradermal comparative test; 
(b) intradermal comparative test for the establishment and maintenance 
of officially tuberculosis-free holding status : 
pos i t i ve a positive bovine reaction which is more than 4mm 
greater than the avian reaction, or the presence of 
cl inical signs; 
inconclusive : a positive or inconclusive bovine reaction which is 
from 1 to 4 mm greater than the avian reaction, and 
the absence of clinical signs; 
negat ive a negative bovine reaction, or a positive or 
inconclusive bovine reaction but which is equal to or 
less than a positive or inconclusive avian reaction 
and the absence of clinical signs in both cases. 
Animals inconclusive to the intradermal comparative test shall be 
subjected to another test after a minimum of 42 days. Animals which 
are not negative to this second test shall be deemed to be positive to 
the test; 
(c) Officially tuberculosis-free holding status may be suspended and 
animals from the holding shall not be allowed to enter intra­
community trade until such time as the status of the following 
animals is resolved: 
1. Animals which have been deemed to be inconclusive to the single 
intradermal tuberculin test; 
2. Animais which have been deemed to be positive to the single 
intradermal tuberculin test but are awaiting retest with an 
intradermal comparative test; 
3. Animals which have been deemed to be inconclusive to the 
intradermal comparative test. 
(d) Where animals are required by Community legislation to be subjected 
to an Intradermal test prior to movement, the test shall be 
interpreted so that no animal which shows an increase in skin fold 
thickness greater than 2mm or the presence of clinical signs is 
entered into intracommunity trade. 
ANNEX C 
BRUCELLOSIS 
A. Serum agglutination tests 
1. The standard agglutinating serum must conform to the standard serum 
prepared by the Veterinary Laboratory, Weybridge, Surrey, England. 
The ampoule must contain 1 000 iu of agglutination obtained by 
lyophiIizing 1 ml of bovine serum. 
2. The standard serum must be that supplied by the Bundesgesundheitsamt, 
Berlin. 
3. The degree of brucella agglutination in a serum must be expressed in iu 
per ml (i.e. Serum x - 80 iu/ml). 
4. Readings of slow sero-agglut inat ion in tubes must be taken at 50 or at 
75% agglutination, the antigen used having been titrated under identical 
conditions against the standard serum. 
5. The agglutinating value of various antigens in relation to standard 
serum must be within the following limits : 
- if the reading is made at 50 % : between 1/600 and 1/1000, 
- if the reading is made at 75 % : between 1/500 and 1/750. 
6. Weybridge strain no 99 and USDA 1119 or any other strain of equivalent 
sensitivity must be used for preparing the antigen for use in tube 
agglutination (slow method). 
7. The culture media used for keeping the strain in the laboratory and for 
producing the antigen must be such that they do not encourage bacterial 
dissociation (s minus r); potato agar should preferably be used. 
8. The bacterial emulsion must be made from physiological saline (NaCI 
8,5%) phenolized at 5 %. Formol must not be used. 
9. The official institutes indicated below must be made responsible for the 
official testing of antigens : 
(a) Germany: Bundesgesundheitsamt, uerlin; 
(b) Belgium: Institut national de recherches vétérinaires, Brussels; 
(c) France: Laboratoire central de recherches vétérinaires, Alfort; 
(d) Grand Duchy of Luxembourg : Institute of the supplying country; 
(e) Italy: Istituto superiore di sanita, Rome; 
(f) Netherlands : Centraal Diergeneeskundig Instituut, Afde ling, 
Rotterdam 
(g) Denmark: Statens Veterinaere Serum laboratorium, Copenhagen v.; 
(h) Ireland: Veterinary Research Laboratory, Department of Agriculture 
and Food, Dub lin; 
(i) United Kingdom : 
- Great Britain : the Central Veterinary Laboratory, 
Weybridge,Surrey, England, 
- Northern Ireland : Veterinary Research Laboratory, 
Stormont.Belfast. 
(J) Greece 
(k) Spain : Centro Nacional de brucelosis de Murcia 
(I) Portugal: Laboratoria Nacional de Investigacao Veterinaria - Lisboa 
10. Antigens may be delivered in the concentrated state provided the 
dilution factor to be used is indicated on the bottle label. 
11. In order to carry out a sero-agglut inat ion test, at least three 
dilutions must be prepared for each serum. Dilutions of suspect serum 
must be made in such a way that the reading of the reaction at the 
infection limit is made in the median tube. If there is a positive 
reaction in this tube, the suspect serum contains at least 30 iu of 
agglutination per ml. 
B. Complement fixation reaction test 
1. The standard serum is the same as that under A.1 of this Annex. In 
addition to its content in international agglutinating units, 1 ml of 
this lyophilized bovine serum must contain 1 000 sensitizing units which 
fix the complement. These sensitizing units are called EEC sensitizing 
units. 
2. The standard serum must be supplied by the bundesgesundheitsamt, Berlin. 
3. A serum's level of antibodies which fix the complement must be expressed 
in EEC sensitizing units (for example : serum x - 60 EEC sensitizing 
units per ml). 
4. A serum containing 20 or more EEC sensitizing units (i.e. An activity 
equal to 20 % of that of the standard serum) per ml, must be considered 
to be pos i t i ve. 
5. Serums must be inactivated as follows : 
(a) bovine serum : 56 to 60*C for 30 to 50 minutes; 
(b) swine serum : 60'C for 30 to 50 minutes. 
6. Weybridge strain no 99 or USDA strain 1119 must be used for the 
preparation of the antigen. The antigen represents a bacterial 
suspension in a physiological serum at 0,85 % or in a veronal loading 
so lut ion. 
7. In order to carry out the reaction test a complementary dose higher than 
the minimum necessary for total haemolysis should be used. 
8. In carrying out the complement fixation reaction test, the following 
controls must be made each time : 
(a) control of the anti-complementary effect of the serum; 
(b) control of the antigen; 
(c) control of sensitized red blood corpuscles; 
(d) control of the complement; 
(e) control using a positive serum of sensitivity at the start of the 
react ion; 
(f) control of the specificity of the reaction using a negative serum. 
9. The supervision and official control of standard serums and antigens 
shall be carried out by the bodies listed in a 9 of this Annex. 
10. Antigens may be delivered in the concentrated state provided the 
dilution factor to be used is indicated on the bottle label. 
C. Ring test 
1. The ring test must be made on the contents of each milk churn or on the 
contents of each bulk tank from the farm. 
2. The standard antigen to be used must come from one of the institutes 
listed in paragraph a.9 (a) to (J). It is recommended that the antigens 
should be standardized according to the WH0/FA0 recommendations. 
3. The antigen may be stained only with haematoxylin or tetrazolium; 
haematoxylin should preferably be used. 
4. If no preservation is used then the reaction test must be carried out 
between 18 and 24 hours of taking the sample from the cow. If milk is to 
be tested later than 24 hours after sampling, then preservation must be 
used, formalin or mercuric chloride may be used as preservatives and if 
either of these are used the test must be carried out within the 
following 14 days after the day of sampling. Formalin may be added to 
give a final concentration in the milk sample of 0,2 % and, in such 
cases, the ratio between the amount of milk and the solution of formalin 
must be at least 10 to 1. A solution of mercuric chloride may be used 
instead of formalin to give a final concentration in the milk of 0,2 % 
and, in such cases, the ratio between the amount of milk and the 
solution of mercuric chloride must be 10 to 1. 
5. The reaction must be carried out using one of the following methods : 
- on a column of milk at least 25 mm high and on a volume of milk of 1 
ml to which 0,03 ml of one of the standardized stained antigens has 
been added, 
- on a column of milk at least 25 mm high and on a volume of milk of 1 
ml to which 0,05 ml of one of the standardized stained antigens has 
been added, 
- on a volume of milk of 8 ml which 0,08 ml of one of the standardized 
stained antigens has been added, 
- on a column of milk at least 25 mm high and on a volume of milk of 2 
ml to which 0,05 ml of one of the standardized stained antigens has 
been added. 
6. The mixture of milk and antigens must be incubated at 37'C for not less 
than 45 minutes and not more than 60 minutes. The test must be assessed 
within 15 minutes of removal from the incubator. 
7. The reaction must be assessed according to the following criteria : 
(a) negative reaction : coloured milk, colourless cream; 
(b) positive reaction : milk and cream identically coloured or 
colourless milk and coloured cream. 
D. The buffered brucella antigen test 
The buffered brucella antigen test may be carried out using one of the 
fol lowing methods : 
a. Manual test 
1. The standard serum shall be the second international standard anti-
brucella abortus serum which is supplied by the Central Veterinary 
Laboratory, Weybridge, Surrey, England. 
2. The antigen shall be prepared without reference to the cell 
concentration, but its sensitivity must be standardized in relation to 
the second international standard anti-brucella abortus serum in such a 
way that the antigen produces a positive reaction with serum dilution of 
1 : 47.5 and a negative reaction with a dilution of 1 : 55. 
3. The antigen shall be suspended in buffered brucella antigen diluent at a 
ph of 3.65 more or less 0.5 and may have been stained by the use of 
rose bengal dye. 
4. Weybridge strain no 99 or usda 1119 or any other strain of equivalent 
sensitivity must be used for preparing the antigen. 
5. The culture media used for keeping the strain in the laboratory and for 
producing the antigen must be such that they do not encourage bacterial 
dissociation (s - r); potato agar medium or continuous culture methods 
should be used. 
6. The antigen shall be tested against eight freeze-dried known positive 
and negative sera. 
7. The official supervision and control of standard serum and antigen shall 
be carried out by the official bodies listed in Annex C (a) (9). 
8. The antigen shall be delivered ready for use. 
•q-h-
9. The buffered brucella antigen test shall be carried out in the following 
manner : 
(a) one drop (0.03 ml) of antigen should be placed alongside one drop 
(0.03 ml) of the serum on a white plate; 
(b) they should be mixed with an applicator stick, first in a straight 
line and then in a circle of about 10 to 12 mm diameter; 
(c) the plate should then be rocked back and forth for four minutes 
(about 30 times per minute); 
(d) readings should be taken in a good light; if there is no evidence of 
agglutination, the test shall be regarded as negative; any degree of 
agglutination shall be regarded as positive, unless there has been 
excessive drying round the edges. 
b. Automated method 
The automated method must be at least as sensitive and accurate as the 
manual method. 
E. Plasma ring-test 
A. extraction of the plasma 
The tube containing blood, coagulation of which having been inhibited by 
the addition of edta, should be centrifuged for three at 3 000 r/min and 
subsequently kept at 37"C for 12 to 24 hours. 
B. Evaluation 
0.2 ml of stabilized plasma should be placed in a tube with 1 ml of 
untreated milk. After mixing, one drop (0.05 ml) of abr-antigen should be 
added and the whole again mixed. The antigen should be standardized in 
relation to a standard antigen supplied by the body referred to in (a) (9) 
(a). 
Following an incubation period of 45 minutes at 37'C, a reading should be 
taken within 15 minutes. The result shall be regarded as positive if the 
colour of the ring has become the same as, or darker than, that of the milk 
coIumn. 
F. Plasma agglutination 
The plasma extracted in accordance with e (a) may be used immediately after 
centrifuging, no thermal stabilization being necessary. 0.05 ml of plasma 
should be mixed with 1 ml of antigen for 50 % sero-agglut inat ion, which 
corresponds to a dilution of 1 : 20 for sero-agglutination. A reading 
should be taken after 18 to 24 hours incubation at 37?C. 50% or more 
agglutination shall be regarded as positive. 
G. Micro-agglutination test 
1. Diluents are made up of 0,85 % physiological saline solution phenolized 
at 0,5 %. 
2. The antigen shall be prepared as described under points 6, 7 and 8 of 
Annex C (a) and shall be titrated as described under point 5 of Annex C 
(a). At the moment the antigen is used safranin o shall be added at 0,02 
% (final dilution). 
3. The standard serum is the same as that under point 1 of Annex C (a). 4. 
The standard serum must be supplied by the bundesgesundheitsamt, berlin. 
5. The micro-agglutination test shall be carried out on plates bearing 
wells with conical bottoms of a volume of 0,250 ml. The test shall be 
carried out as follows : 
(a) predilution of the serum : 0,050 ml of each serum to be tested are 
added to each well containing 0,075 ml of diluent. The mixtures are 
shaken for 30 seconds. 
(b) gradual serum dilution : prepare at least three dilutions for each 
serum, to this end from the predilutions (1 : 2,5) one takes 0,025 
ml of each serum and transfers them to a well containing 0,025 ml of 
diluent. In this way the first dilution reaches a strength of 1 : 5 
and the following dilutions are carried out by doubling. 
(c) addition of antigen : 0,025 m< of antigen is added to each well 
containing the different serum dilutions. After being shaken for 30 
seconds the plates are closed with their respective lids and kept at 
37'C for 20 to 24 hours in a humidified atmosphere. 
(d) reading the results : assessment of the aspect of the sedimentation 
of the antigen is made by examining the bottom of the well reflected 
in a concave mirror placed above it. If there is a negative 
reaction, the antigen forms a sediment in the form of a compact 
button with clear edges and having an intense red colour. If there 
is a positive reaction, on the other hand, a diffused pink veil is 
formed that is evenly distributed. The different percentages of 
agglutination are determined by comparison with antigen checks 
indicating 0, 25, 50, 75 and 100 % agglutination. The title of each 
serum is expressed in international units of agglutination per ml. 
There should be included in the test, controls with negative and 
positive serum diluted so as to contain 30 international units of 
agglutinât ion per ml. 
H. Enzyme-1 inked immunosorbent assay (Eli sa) for detecting bovine brucellosis. 
1. The material and reagents to be used are as follows: 
(a) solid phase microplates, cuvettes or any other solid phase; 
(b) the antigen is fixed to the solid phase with or without the aid of 
polyclonal or monoclonal catching antibodies. 
(c) the biological fluid to be tested; 
(d) a corresponding positive and negative control; 
(e) conjugate; 
(f) a substrate adapted to the enzyme used; 
(g) a stopping solution, if necessary; 
(h) solutions for the dilution of che test samples for preparations of 
the reagents and for washing; 
(i) a reading system appropriate to the substrate used. 
2. Standardization and sensitivity of test: 
1. bulk milk samples are classified negative if they give a reaction 
less than 50 % of that given by a 1 in 10 000 dilution of the second 
international brucellosis standard serum made up in negative milk; 
2. individual serum samples are classified negative if they give a 
reaction less than 10 % of that given by a 1 in 200 dilution of the 
second international brucellosis standard serum made up in saline 
solution or in any other recognized dilution, in accordance with the 
procedure laid down in Article 12 after receiving the opinion of the 
Scientific Veterinary Committee. 
The brucellosis Elisa standards shall be as specified in Annex C, A. 1 
and A.2 (to be used at the dilutions indicated on the label). 
3. Conditions for use of the Elisa test for bovine brucellosis 
The Elisa method may be used on a sample of milk or whey taken from the 
milk collected from a farm with at least 30 % of dairy cows in milk. 
If this method is used, 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. 
~ H r 
ANNEX D 
CHAPTER I 
ENZOOTIC BOVINE LEUCOSIS FREE HOLDINGS. MEMBER STATES AND REGIONS 
A. A holding is an Enzootic Bovine Leucosis Free holding if: 
(i) there is no evidence, either clinical or as a result of a laboratory 
test, of any case of enzootic bovine leucosis in the holding and no 
such case has been confirmed in the previous two years, and 
(ii) all animals over 24 months of age have reacted negatively during the 
preceding 12 months to two tests carried out in accordance with this 
Annex, at an interval of at least four months, or 
(Hi) it meets the requirements of (i) above and is situated in an 
enzootic bovine leucosis free Member State or region. 
B. An individual holding shall retain Enzootic Bovine Leucosis Free status if: 
(') the condition In paragraph A(I) continues to be fulfilled. 
( * i ) any animals introduced into the holding must come from an enzootic 
bovine leucosis free holding 
(iii) all animals over 24 months of age continue to react negatively to a 
test carried out in accordance with Chapter M at intervals of three 
y$ars 
C. The Leucosis Free Status of a holding shall be suspended if the conditions 
detailed in B above have not been complied with. 
D. The status shall remain suspended until the following requirements are 
complied with: 
1. If a single animal in an enzootic bovine leucosis free holding has 
reacted positively to one of the tests referred to in Chapter II: 
<*6' 
(i) the animal which has reacted positively, and, in the case of a 
cow, any calf it may have produced, must have left the holding 
for slaughter under the supervision of the veterinary 
authorities; 
(ii) the remaining animals have reacted negatively to a serological 
test carried out in accordance with Chapter II three months at 
least after removal of the positive animal and any possible 
progeny thereof; 
(Mi) an epidemiological enquiry must be conducted and the holdings 
linked epidemiologically to the infected holding must be 
subjected to the measures laid down in (ii); 
However, the competent authority may grant a derogation from the 
obligation to slaughter the calf of an infected cow where it was 
separated from its mother after calving. In this case, the calf must be 
made subject to the requirements provided for in 2 (iii) below. 
2. Where more than one animal from an Enzootic Bovine Leucosis Free holding 
has reacted positively or where infection has been confirmed in a 
ho IdIng: 
(i) the animals which have reacted positively and their calves, in 
the case of cows, must be removed for slaughter under the 
supervision of the veterinary authorities; 
(ii) all animals aged over 24 months must react negatively to two 
tests carried out in accordance with Chapter II at an interval of 
at least 4 months and less than 12 months; 
(iii) all other animals must, after identification, remain on the 
holding until they are aged over 24 months and have satisfied the 
tests referred to in (ii) above. 
(iv) an epidemiological enquiry must be conducted, and the holdings 
linked epidemiologically to the infected holding must be 
subjected to the measures laid down in (ii) above. 
^ 
However, the competent authority may grant a derogation from the 
obligation to slaughter the calf of an infected cow where it was 
separated from its mother after calving. In this case, the calf must be 
made subject to the requirements provided for in 2(iii). 
3. Where the Enzootic Bovine Leucosis Free status of a holding has been 
suspended for any other reason, al. animals in the holding aged over 24 
months must give a negative reaction to a serological test carried out 
In accordance with Chapter II. 
E. In accordance with the procedure in Article 12. the Commission may propose 
that a Member State or Region of a Member State may become Enzootic Bovine 
Uucosis Fre$ if: 
(a) at least 99,8 % of the bovine holdings are enzootic bovine leucosis 
free holdings within the meaning of A above, 
or 
(b) no case of enzootic bovine leucosis has been confirmed in the Member 
State or region for the past three years and 
in the case of a Member State, all animals aged over 24 months in at 
least 10% of herds, selected randomly, have been tested with negative 
results in accordance with Chapter II in the previous 24 months or 
in the case of a region or a Member State, all animals aged over 24 
months have undergone a test provided for in Chapter II with negative 
result 
F. A Member State or a Region of a Member State shall retain Enzootic Bovine 
Leucosis Free status if: 
(i) every year either a random sample with a confidence rating of 99% has 
established that less than 0,2% of the holdings were infected or not 
less than 20% of bovine animals over two years of age have been tested 
and have reacted negatively to a test carried out in accordance with 
Chapter 11. 
or 
(ii) where no case of enzootic bovine leucosis has been recorded in the 
Member State or Region in a proportion of one holding out of 10 000 
for at least three years, a decision may be taken in accordance with 
Article 12 to cease routine serological testing provided that; 
- all cattle slaughtered within the territory of that Member State or 
region are submitted to a post mortem examination by an official 
veterinarian who must issue notification of all tumours with a view to 
laboratory examination, and. 
- the Member state shall report the occurrence of all cases of enzootic 
bovine leucosis in the area affected by the decision to the 
Commission. The Commission may according to the procedure in Article 
12 propose that the decision to cease routine serological testing be 
suspended or revoked and 
- any cattle which react positively to an immune-diffusion test are 
slaughtered and the holding remains subject to restrictions until re-
establishment of its status pursuant to Annex D, Chapter I, D. 
G. (i) The Enzootic Bovine Leucosis Free status of a Member State or Region 
of a Member state shall be suspended, in accordance with the procedure 
in Article 12. if enzootic bovine leucosis is detected and confirmed 
in more than 0.2% of holdings in the region or Member State. 
(i i) The Enzootic Bovine Leucosis Free status may be restored, in 
accordance with the procedure in Article 12. if; 
a. in addition to the measures provided for in paragraphs D.1 and D.2 
above, at least 20% of the other holdings, selected randomly, in the 
region or Member State have, within a 12 month period, undergone one 
of the tests referred to in Chapter II. 
b. the results of this testing establish, with a confidence rating of 
99%. that not less than 0.2% of holdings are infected. 
_ -) / -
ANNEX D 
CHAPTER II 
TESTS FOR ENZOOTIC BOVINE LEUCOSIS 
Tests for enzootic bovine leucosis shall be carried out by the immune-
diffusion test under the conditions described in points A and B below or by 
the enzyme-linked immunosorbent assay (biisa) under the conditions described 
in point C below. The immune-diffusion method may only be used for individual 
tests. If test results are the subject of a duly-substantiated challenge, an 
additional check shall be carried out by means of the 
immune-diffusion test. 
A. Agar gel immune-diffusion test for enzootic bovine leucosis 
1. The antigen to be used in the test must contain bovine leucosis virus 
glycoproteins. The antigen must be standardized against a standard serum 
(El serum) supplied by the State Veterinary Serum Laboratory, 
Copenhagen. 
2. The official institutes indicated below must be made responsible for 
calibrating the standard working antigen of the laboratory against the 
official EEC standard serum (El serum) provided by the State Veterinary 
Serum Laboratory, Copenhagen. 
(a) Germany: Bundesforschungsanstalt fur Viruskrankheiten der Tiere, 
Tub i ngen 
(b) Belgium: Institut national de recherches vétérinaires, Bruxelles 
(c) France: Laboratoire national de pathologie bovine, Lyon 
(d) Grand Duchy of Luxembourg:— 
'i/ 
(e) Italy: Istituto ZooprofiI attico Spenmentale, Perugia 
(f) Netherlands: Centraal Diergeneeskundig Instituut, Afdeling Rotterdam 
(g) Denmark: Statens Veterinaere Serum Laboratorium, Copenhagen 
(h) Ireland: Veterinary Research Laboratory, Abbotstown, Dublin 
(i) United Kingdom: 
1. Great Britain: The Central Veterinary 
Weybridge,Engl and Laboratory, 
2. Northern Ireland: The Veterinary Research Laboratory, Stormont, 
Belfast 
(J) Spain: Subdirreccion general de sanidad animal. 
Laboratorio de sanidad y produce ion animal ALGETE 
(Madrid); 
(k) Portugal : 
(I) Greece: Laboratorio Nacional de Investigacao Veterinaria, 
Lisboa 
3. The standard antigens used in the laboratory must be submitted at least 
once a year to the EEC reference laboratories listed in paragraph 2 above 
for testing against the official EEC standard serum. Apart from this 
standardization the antigen in use can be calibrated in accordance with B. 
4. The reagents for the test shall consist of: 
(a) antigen: the antigen must contain specific glycoproteins of enzootic 
bovine leucosis virus which has been standardized against the official 
EEC serum; 
(b) the test serum; 
(c) known positive control serum; 
(d) Agar gel, 
0,8% agar, 
8,5% NaCI, 
0,OS M Tris-buffer pH 7,2, 
15 ml of this agar must be introduced into a petri dish of 85 mm 
diameter, resulting in a depth of 2,6 mm of agar. 
5. A test pattern of seven moisture-free wells be cut in the agar to the 
bottom of the plate; the pattern must consist of one central well and six 
wells in a circle around it. 
Diameter of central well: 4 mm 
Diameter of peripheral wells: 6 mm 
Distance between central and peripheral wells: 3 mm 
6. The central well must be filled with the standard antigen.The peripheral 
wells 1 and 4 (see diagram below) are filled with the known positive serum, 
the wells 2, 3, 5 and 6 with the test sera. The wells must be filled until 
the meniscus disappears. 
i 
O 
6 O O 2 
o 
SO 03 
o 
4 
'•» H-
7. This results in the following quantities being obtained: 
antigen: 32 fi\ 
control serum: 73 fi\ 
test serum: 73 fi\ 
8. Incubation must be for 72 hours at room temperature (20 to 27*C) in a 
closed humid chamber. 
9. The test may be read at 24 and 48 hours but a final result may not be 
obtained before 72 hours: 
(a) a test serum is positive if it forms a specific precipitin line with 
the BLV antigen and forms a complete line of identity with the control 
serum; 
(b) a test serum is negative if it does not form a specific precipitin 
line with the BLV antigen and if it does not bend the line of the 
control serum; 
(c) the reaction cannot be considered conclusive if it: 
(i) bends the line of the control serum towards the BLV antigen well 
without forming a visible precipitin line with the antigen; 
or 
(ii)if it cannot be read either as negative or as positive. 
In inconclusive reactions the test may be repeated and concentrated serum 
ut iIized. 
10. Any other well configuration or pattern may be utilised provided that 
the E4 serum diluted 1:10 In negative serum, can be detected as 
POS i 11ve. 
- rs-
B. Method for antigen standardization 
Solutions and materials required: 
1. 10 ml of 1,6% agarose in 0,05% M Tns/HCI buffer, pH 7,2 with 8,5% NaCI. 
2. 15 ml of a bovine leucosis serum, having antibody only to bovine 
leucosis virus glycoproteins, diluted 1:10 in 0,05 M Tris/HCI buffer, pH 
7,2 with 8,5% NaCI. 
3. 15 ml of a bovine leucosis serum, having antibody only to bovine 
leucosis virus glycoproteins, diluted l:5 in 0,05 M Tris/HCI buffer, pH 
7,2 with 8,5% NaCI. 
4. Four plastic petri dishes with a diameter of 85 mm. 
5. A punch with a diameter of 4 to 6 mm. 
6. A reference antigen. 
7. The antigen which is to be standardized. 
8. A water bath (56 *C). 
Procedure: 
Dissolve the agarosed,6%) in the Tris/HCI buffer by carefully heating to 
lOO'C. Place in 56 *C waterbath for approximately one hour. Also, place the 
bovine leucosis serum dilutions in 56'C water bath. 
Now, mix 15 ml of the 56'C agarose solution with the 15mI bovine leucosis 
serum (1:10),quickly shake and pour 15 ml into each of two petri dishes. 
Repeat this procedure with the bovine leucosis serum diluted 1:5. 
When the agarose has hardened, holes are made in it as follows: 
6<É-
Petri dish No 1 
Serum 1:10 Petri dish No 2 
Scrum 1:10 
Petri dish No 3 
Serum 1:5 Petri dish No 4 
Serum 1:5 
Add i t i on of ant i gen : 
(i). Petri dishes 1 and 3 
well A - undiluted reference antigen, 
well B - 1:2 diluted reference antigen, 
wells C and E - reference antigen, 
well D « undiluted antigen to be tested 
(i i). Petri dishes 2 and 4 
well A - undiluted test antigen, 
well B « 1:2 diluted test antigen, 
well C » 1:4 diluted test antigen, 
well D - 1:8 diluted test antigen. 
^ 
Additional Instructions: 
1. The experiment shall be carried out with two serum dilutions (1:5 and 
1:10) in order' to achieve optimal precipitation. 
2. If the precipitation diameter Is too small with both dilutions, then the 
serum must be further diluted. 
3. If the precipitation diameter in boch dilutions is too large and faint, 
then a lower serum must be chosen 
4. The final concentration of the agarose must be 0,8%; that of the sera 5% 
and 10% respectively. 
5. Plot the measured diameters In the following coordinate system. The 
dilution of the antigen to be tested with the same diameter as the 
reference antigen is the working dilution. 
Diameter 
13 -
12 -
11 -
10 -
9 — 
8 -
7 — 
6 -
5 -
4 — 
3 -
2 -
1 -/ 
' ' 1:3 I 1:5 1:6 1:7 I 
1:1 1:2 1:4 1:8 
Dilutions of anrigens 
">% 
C. Enzvme-linked immunosorbent assay (Elisa) for detecting enzootic bovine 
ieucosis. 
1. The material and reagents to be used are as follows: 
(a) solid phase microplates, cuvettes or any other solid 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 
EBL. The control antigen should be identical to the antigen except 
for the BLV antigens. If catching antibodies are coated to the solid 
phase the antibodies must not react to antigens other than BLV 
antigens; 
(c) the biological fluid to be tested; 
(d) a corresponding positive and negative control; 
(e) conjugate; 
(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; 
(i) a reading system appropriate to the substrate used. 
S3-
2. Standardization and sensitivity of test: 
The sensitivity of the Elisa 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 official institutes indicated in point A.2 will be 
responsible for checking the quality of the Elisa method, and in 
particular to determine, for each production batch, the number of 
samples to be pooled on the basis of the count obtained for the E4 
serum. 
The E4 serum will be supplied by the National Veterinary Laboratory, 
Copenhagen. 
3. Conditions for use of the Elisa test for EBL 
The Elisa method may be used on a sample of milk or whey taken from the 
milk collected from a farm with at least 30 % of dairy cows in milk. 
If this method is used, 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. 
ANNEX E(l) 
(a) bovine diseases : 
- Rabies. 
- Tuberculosis. 
- Brucellosis, 
- Contagious Bovine Pleuropneumonia. 
- Enzoot i c Bov i ne Leucos i s 
(b) swine diseases 
- Rabies. 
- Bruce 11osis 
ANNEX EM I) 
Auieszky's Disease 
Infectious Bovine Rhinotracheitis 
Brucella suis infection 
CM-
CERT NO SPECIES bovine/swine for slaughter/breeding/production 
MEMBER STATE of origin REGION OF ORIGIN 
HOLDING OF ORIGIN 
Name 
ref number 
The holding of origin is; 
officially tuberculosis free 
officially brucellosis free 
Brucellosis free 
leucosis free. 
DATE OF DEPARTURE 
Signed yes/no 
yes/no 
yes/no 
yes/no address 
The a. imals listed below have been tested in 
compliance with Directive 64/432/EEC as 
follows; 
TEST DATE 
tuberculin test yes/not required . . 
brucellosis SAT yes/not required . . 
leucosis test yes/not required . . 
Official veterinarian, holding of origin 
ANIMAL IDENTIFICATION 
Breed type age total animals 
official identification 
APPROVED MARKET 
Location 
Address 
date ref no 
Sian^ture/stamD LOADING/ASSEMBLY POINT 
Location 
Address 
date ref no 
additional guarantees for 
to (Com. Decision 
date health examination 
expiry date of this cert INTRACOMMUNITY TRADE 
Means of transport 
Identification 
Following due enquiry, I certify that (1) all 
I applicable provisions of Council Directive 
64/432/EEC have been complied with. 
(2) The proposed movement has been registered 
Ion ANIMO. 
(3)The animal(s) listed above comply with the 
disease for (species/type) destined 
/ /EEC) 
signed date 
Name block capitals 
ANNEX I I 
CORRELATION TABLE 
Updated Directive Directive 64/432/EEC 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 6 1 
e 2.(a) 
e 2.(b) 
e 2.(c) 
e 2.(d) 
e 2.(e) 
e 2.(f) 
e 2.(g) 
e 2.(h) 
e 2.(i) 
e 2.(J) 
e 2.(k) 
e 2.(1) 
e 3.1 
e 3.2.(a) 
e 3.2.(b) 
e 3.2.(c) 
e 3.2.(d) 
e 3.2.(e) 
e 4.1. 
e 4.2 
e 4.3. 
e 5 
e 6.1, first indent 
e 6.1, second indent 
e 6.2.(a), 1st sub­
paragraph 
e 6.2.(a), 2nd sub­
paragraph 
e 6.2.(b), 1st sub­
paragraph 
e 6.2.(b), 2nd sub­
paragraph 
6.2.(c) 
6.2.(d) 
6.3. 
7 
8 
9 
10 
11 
12.1. 
12.2. 
12.3. Article 1 
Article 2.(b) 
Article 2.(c) 
Article 2.(d) 
Article 2.(e) 
Article 2.(f) 
Article 2.(s) 
Article 2.(t) 
Article 2.(1) 
Article 3.1 
Article 3.2.(a) 
Article 3.2.(b) 
Article 3.2.(e) 
Article 3.5 
Article 3.2.(f)(i) 
Article 3.2.(g) 
Article 3.2.(d) 
Article 3.3.(a) 
Article 3.3.(b) 
Article 3.3.(d) 
Article 3.2.(f)(ii) 
Article 6 
Article 9 
Article 10 
Article 12.1. 
Article 12.2. 
Article 12.3. 
^°>-
Updated Direct ive 
Article 12.4., 1st sub­
paragraph 
Article 12.4., 2nd sub­
paragraph 
Article 12.4., 3rd sub­
paragraph 
Article 13.1. 
Article 13.2. 
Article 13.3. 
Article 13.4., 1st sub­
paragraph 
Article 13.4., 2nd sub­
paragraph 
Article 14 
Article 15 
Annex A 
Annex B.1.- B.26. 
Annex B.27. 
Annex B.28.- B.31. 
Annex B.32. 
Annex C.A. 
Annex C.B. 
Annex C.C. 
Annex CD. 
Annex C.E. 
Annex C.F. 
Annex C.G. 
Annex C.H. 
Annex D.I. 
Annex D.I I.A.1 - D.I I.A.9 
Annex D.11.A.10 
Annex D.11.B. 
Annex D.11.C. 
Annex E.I. 
Annex E.11. 
Annex F. Directive 64/432/EEC 
Article 12.4., 1st sub­
paragraph 
Article 12.4., 2nd sub­
paragraph 
Article 13.1. 
Article 13.2. 
Article 13.3. 
Article 13.4., 1st sub­
paragraph 
Article 14 
Article 16 
Annex B.1.- B.26. 
Annex B.27. 
Annex B.28.- B.31. 
Annex B.32 
Annex C.A. 
Annex C.B. 
Annex C.C. 
Annex CD. 
Annex CE. 
Annex CF. 
Annex C.G. 
Annex G.11.C 
Annex G.I. 
Annex G.11.A.1 - G.I I.A.9 
Annex G.I I.B. 
Annex G. 11 .C 
ISSN 0254-1475 
COM(93) 698 final 
DOCUMENTS 
EN 10 03 
Catalogue number : CB-CO-93-750-EN-C 
ISBN 92-77-63141-4 
Office for Official Publications of the European Communities 
L-2985 Luxembourg