CELEX: 51993PC0698
Language: en
Date: 1994-01-07
Title: Proposal for a COUNCIL DIRECTIVE amending and updating Directive 64/432/EEC on health problems affecting intra- Community trade in bovine animals and swine

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                                 COMMISSION OF THE EUROPEAN COMVTUNITIES
                                                                         C0M(93) 698 final
                                                                         Brussels, 7 January 1994
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                                                          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)
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 ---pagebreak---                                      -   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.
 ---pagebreak---                                          - 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
                                                           7
specific measures relating to swine vesicular disease ;
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
 ---pagebreak---                                          - 2 -
Whereas   it is necessary,    in the light of this situation, to modify Council
Directive 64/432/EEC 8 of 26 June 1964 on health problems affecting          intra-
Community    trade  in bovine animals and swine, as       last amended  by  Council
Directive 92/102/EEC 9 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
 ---pagebreak---                                      - 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.;
 ---pagebreak---                                       - 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;
 ---pagebreak---                                       - 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.
 ---pagebreak---                                           - 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 :
 ---pagebreak---                                          - 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.
 ---pagebreak---                                            - 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.
 ---pagebreak---                                          - 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.
 ---pagebreak---                                        - 10 -
                                     Art icle 8
Any person or persons suspecting the presence of one of the diseases listed in
Annex E d ) 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 E M I ) 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,
 ---pagebreak---                                         - 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 E M I ) , 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,
 ---pagebreak---                                        - 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.
 ---pagebreak---                                       - 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.
 ---pagebreak---                                      - 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 1992 1 0 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
 ---pagebreak---                                      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
 ---pagebreak---                                       - <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.
 ---pagebreak---                                             -  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.
 ---pagebreak---                                            ^
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.
 ---pagebreak---                                            ' *\
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
 ---pagebreak---                                                  - cJtt-
    ( i i)     t h r e e m i l k E L I S A ' s c a r r i e d o u t at i n t e r v a l s o f 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:
 ---pagebreak---         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.
 ---pagebreak---                                         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.
 ---pagebreak---                                           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.
 ---pagebreak---                                       — 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.
 ---pagebreak---                                         -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
 ---pagebreak---                                            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.
 ---pagebreak---                                      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.
 ---pagebreak---                                         '«
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;
 ---pagebreak---                                        -=^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.
 ---pagebreak---                                               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.
 ---pagebreak---                                        •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
 ---pagebreak---           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.
 ---pagebreak--- 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
 ---pagebreak---         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).
 ---pagebreak---   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;
 ---pagebreak---     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.
 ---pagebreak---                                       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.
 ---pagebreak--- 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.
 ---pagebreak---    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 m l ) .
   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.
 ---pagebreak---    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
 ---pagebreak---    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.
 ---pagebreak--- 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.
 ---pagebreak---                                        •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).
 ---pagebreak---    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.
 ---pagebreak---       (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;
 ---pagebreak---    (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.
 ---pagebreak---                                        ~ Hr
                                      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:
 ---pagebreak---                                          <*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.
 ---pagebreak---                                         ^
      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
 ---pagebreak---    (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.
 ---pagebreak---                                         _ -) / -
                                      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:—
 ---pagebreak---                                           '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        Laboratory,
                               Weybridge,Engl and
       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 :              Laboratorio Nacional de Investigacao Veterinaria,
                               Lisboa
   (I) Greece:
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;
 ---pagebreak---    (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
                         6O               O 2
                                   o
                         SO               03
                                   o
                                    4
 ---pagebreak---                                           '•» 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.
 ---pagebreak---                                       - 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:
 ---pagebreak---                                                             6<É-
Petri dish No 1                                 Petri dish No 2
Serum 1:10                                      Scrum 1:10
 Petri dish No 3                                 Petri dish No 4
 Serum 1:5                                       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.
 ---pagebreak---                                                  ^
           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
 ---pagebreak---                                          ">%
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.
 ---pagebreak---                                             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.
 ---pagebreak---                                   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 E M I)
     Auieszky's Disease
      Infectious Bovine Rhinotracheitis
     Brucella suis infection
 ---pagebreak---                                                CM-
CERT NO             SPECIES bovine/swine    for    slaughter/breeding/production
MEMBER STATE of origin                      REGION OF ORIGIN
HOLDING OF ORIGIN
                                            address
Name
ref number
The holding of origin is;                   The a. imals listed below have been tested in
officially tuberculosis free     yes/no     compliance with Directive 64/432/EEC as
officially brucellosis free      yes/no     follows;
Brucellosis free                 yes/no                                          TEST DATE
leucosis free.                   yes/no     tuberculin test     yes/not required    . .
                                            brucellosis SAT     yes/not required    . .
DATE OF DEPARTURE                           leucosis test       yes/not required    . .
Signed                                      Official veterinarian, holding of origin
ANIMAL IDENTIFICATION                       total animals
Breed              type         age         official identification
APPROVED MARKET                             LOADING/ASSEMBLY POINT
Location                                    Location
Address                                     Address
date            ref no                      date                 ref no
Sian^ture/stamD
                                            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
additional guarantees for               disease for                 (species/type) destined
to                   (Com. Decision   /   /EEC)
                                            signed                          date
date health examination
expiry date of this cert                    Name block capitals
 ---pagebreak---                                 ANNEX I I
                            CORRELATION TABLE
Updated Directive                     Directive 64/432/EEC
Art   6 1                             Article 1
Art   e 2.(a)
Art   e  2.(b)                        Article 2.(b)
Art   e  2.(c)                        Article 2.(c)
Art   e  2.(d)                        Article 2.(d)
Art   e  2.(e)
Art   e  2.(f)                        Article 2.(e)
Art   e  2.(g)
Art   e  2.(h)
Art   e  2.(i)                        Article  2.(f)
Art   e  2.(J)                        Article  2.(s)
Art   e  2.(k)                        Article  2.(t)
Art   e 2.(1)                         Article 2.(1)
Art   e 3.1                           Article 3.1
Art   e  3.2.(a)                      Article  3.2.(a)
Art   e  3.2.(b)                      Article  3.2.(b)
Art   e  3.2.(c)                      Article  3.2.(e)
Art   e  3.2.(d)                      Article 3.5
Art   e  3.2.(e)
Art   e 4.1.                          Article 3.2.(f)(i)
Art   e 4.2                           Article 3.2.(g)
Art   e 4.3.
Art   e 5
Art   e 6.1, first indent             Article 3.2.(d)
Art   e 6.1, second indent
Art   e 6.2.(a), 1st sub-             Article 3.3.(a)
                  paragraph
Art   e 6.2.(a), 2nd sub-
                  paragraph
Art   e 6.2.(b), 1st sub-             Article 3.3.(b)
                  paragraph
Art   e 6.2.(b), 2nd sub-
                  paragraph
Art     6.2.(c)                       Article 3.3.(d)
Art     6.2.(d)                       Article 3.2.(f)(ii)
Art     6.3.
Art     7                             Article 6
Art     8
Art     9                             Article 9
Art     10                            Article 10
Art     11
Art     12.1.                         Article 12.1.
Art     12.2.                         Article 12.2.
Art     12.3.                         Article 12.3.
 ---pagebreak---                             ^°>-
Updated Direct ive          Directive 64/432/EEC
Article 12.4., 1st sub-     Article 12.4., 1st sub-
                paragraph                   paragraph
Article 12.4., 2nd sub-     Article 12.4., 2nd sub-
                paragraph                   paragraph
Article 12.4., 3rd sub-
                paragraph
Article 13.1.               Article 13.1.
Article 13.2.               Article 13.2.
Article 13.3.               Article 13.3.
Article 13.4., 1st sub-     Article  13.4., 1st sub-
                paragraph                   paragraph
Article 13.4., 2nd sub-
                paragraph
Article 14                  Article 14
Article 15                  Article 16
Annex A
Annex B.1.- B.26.           Annex B.1.- B.26.
Annex B.27.                 Annex B.27.
Annex B.28.- B.31.          Annex B.28.- B.31.
Annex B.32.                 Annex B.32
Annex C.A.                  Annex C.A.
Annex C.B.                  Annex C.B.
Annex C.C.                  Annex C.C.
Annex C D .                 Annex CD.
Annex C.E.                  Annex CE.
Annex C.F.                  Annex CF.
Annex C.G.                  Annex C.G.
Annex C.H.                  Annex G.11.C
Annex D.I.                  Annex G.I.
Annex D.I I.A.1 - D.I I.A.9 Annex G.11.A.1 - G.I I.A.9
Annex D.11.A.10
Annex D.11.B.               Annex G.I I.B.
Annex D.11.C.               Annex G. 11 . C
Annex E.I.
Annex E.11.
Annex F.
 ---pagebreak---                                                                      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