Proposal for a COUNCIL DIRECTIVE amending and updating Directive 64/432/EEC on health problems affecting intra- Community trade in bovine animals and swine
Date: 1994-01-07
Institutions: European Commission
Subjects: cattle, exchange of information, health control, intra-EU trade, swine, veterinary legislation

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COMMISSION  OF  THE  EUROPEAN  COMVTUNITIES 

C0M(93) 698 final 

Brussels, 7 January  1994 

Proposal  for a 

COUNCIL DIRECTIVE 

amending  and updating Directive 64/432/EEC 

on health problems affecting  intra-Community  trade 

in bovine animals and swine 

(presented  by  the Commission) 

- 

I -

EXPLANATORY  MEMORANDUM 

Recently,  Community  legislation  has  been  adopted  to  control  Foot  and  Mouth 

disease,  Exotic  diseases,  and  African  Swine  Fever.  It  is  therefore  no  longer 

appropriate  to  retain  control  provisions  for  these  diseases  in  Directive 

64/432/EEC  which  deals  with  intra-Community  trade  in  live  pigs  and  bovines. 

It  is  necessary  to  remove  these  sections  from  Directive  64/432/EEC  and  make 

reference  to  the  relevant  control  Directive. 

In the  light  of  the  Single  Market,  it  is necessary  to  remove  the  requirement 

that  animals  undergo  a  minimum  residency  period  of  six  months  in  a  Member 

State  prior  to  each  movement.  In  addition,  calves  aged  under  15  days  should 

be  brought  within  the  scope  of  the  Directive  and  references  to  frontier  posts 

should  be  removed. 

The  requirement  to  carry  out  premovement  tests  on  animals  moving  from 

countries  or  regions  recognised  as  free  of  Tuberculosis,  Brucellosis  and 

Enzootic  Bovine  Leukosis  and  to carry  out  mastitis  tests  on  dairy  cows  should 

also  be  removed  as a  further  step  towards  the Single  Market. 

The  Directive  has  been  amended  on  over  forty  occasions  since  1964  and  it  is 

now  appropriate  that  it  be  updated.  The  opportunity  has  also  been  taken  to 

simplify  and  clarify  the provisions of  the Directive. 

The  attached  proposal  is designed  to accomplish  these  objectives. 

There  are  no  financial  repercussions on  the Community  budget  involved. 

- va -

Proposal 

for a 

CounciI  Directive 

of 

amending  and updating Directive 64/432/EEC 

on health problems affecting  intra-Community  trade 

in bovine animals and swine 

THE COUNCIL OF THE EUROPEAN  UNION. 

Having  regard  to  the Treaty  establishing  the  European 

Community,  and 

in particular  Article 43, 

Having  regard  to the proposal  from  the  Commission,1 

Having  regard  to the opinion of  the European  Parliament,2 

Having  regard  to the opinion of  the Economic  and Social  Committee,3 

Whereas  important  progress  has  been  made  in  the  harmonisation  of  the veteri 

nary  field;  in particular  the Council  has  adopted  Directive  90/425/EEC  of  26 

June  1990  concerning  veterinary  and  zootechnical  checks  applicable  in 

intracommunity  trade  in certain  live  animals  and  products  with  a  view  to the 

completion  of  the  internal  market4,  Directive  91/496/EEC  of  15  Juiy  >991 

laying  down  the principles governing  the organisation of  veterinary  checks on 

animals  entering  the Community  from  third  countries5, Directive  85/511/EEC of 

18  November  1985  introducing  Community  measures  for  the  control  of  foot  and 

mouth  disease6  and  Directive  92/117/EEC  of  17  December  1992  introducing 

general  Community  measures  for  the  control  of  certain  animal  diseases  and 

specific measures  relating  to swine vesicular  disease7; 

1 OJ  No C 

2 OJ  No C 

3 OJ  No C 

4 OJ No L 224, 18.08.1990, p.29 

5 OJ  No L 268, 24.09.1991, p.56 

6 OJ  No L 315, 26.11.1985, p.11 

7 OJ  No L 62, 15.03.1993, p.69 

-  2 -

Whereas  it  is  necessary,  in  the  light  of  this  situation,  to  modify  Council 

Directive  64/432/EEC8  of  26  June  1964  on  health  problems  affecting  intra-

Community  trade  in  bovine  animals  and  swine,  as  last  amended  by  Council 

Directive  92/102/EEC9  of  5  December  1992,  in particular  concerning  the  period 

of  residence  in  a  Member  State  prior  to  movement,  rules  for  trade  in  animals 

aged  under  15 days  and  rules  for  control  of  certain  diseases; 

Whereas  Directive  64/432/EEC  has  been  substantially  amended  on  several 

occasions;  that  it  is  thus  advisable  for  the  sake  of  clarity  and  rationality 

to bring  the Directive  up-to-date. 

HAS ADOPTED  THIS  DIRECTIVE 

Article 1 

Directive  64/432/EEC  is  hereby  replaced  by  the  text  in  Annex  I  to  this 

Direct ive. 

Article  2 

Member  States  shall  bring  into  force  the  laws,  regulations  and  administrative 

provisions  necessary  to  comply  with  this  Directive  not  later  than  1  July 

1994.  They  shall  forthwith  inform  the Commission  thereof. 

When  Member  States  adopt  these  measures,  they  shall  contain  a  reference  to 

this  Directive  or  shall  be  accompanied  by  such  reference  on  the  occasion  of 

their  official  publication.  The  methods  of  making  such  a  reference  shall  be 

laid  down  by  the Member  States. 

Article  3 

This  directive  is addressed  to  the Member  States 

8  OJ  No  L  121, 29.07.1964,  1977/64 

9  OJ  No L  355, 5.12.1992,  p.32 

-  3 -

ANNEX I 

Article 1 

This  directive  shall  apply  to  intra-community  trade  in  bovine  animals  and 

swine. 

Article 2 

For  the purposes of  this directive : 

(a)  holding  :  shall  mean  a  holding  as  defined  in  Article  2  of  Council 

Directive 90/425/EEC. 

(b)  animal  for slaughter  : 

means  a  bovine  animal  (including  the  species  Bubalus  bubalus)  or 

swine  intended  to be  taken  to a  slaughterhouse  or  market  from  which 

it may only move  to slaughter: 

(c)  animals for breeding or production : 

means  bovine  animals  (including  the  species  Bubalus  bubalus)  and 

swine  other  than  those  referred  to  in  (b), including  those  intended 

for breeding, milk or meat production, or draft purposes; 

(d)  officially  tuberculosis-free bovine holding : 

means  a  bovine  holding  which  satisfies  the  conditions  laid  down  in 

Annex  A.I  1.2 and 3: 

(e)  officially  tuberculosis-free Member  State or part of a Member  State : 

means  a  Member  State  or  part  of  a  Member  State  which  satisfies  the 

conditions  laid down  in Annex A.I  4.5  and  6: 

(f)  officially  brucellosis-free bovine holding : 

means  a  bovine  holding  which  satisfies  the  conditions  laid  down  in 

Annex A  I I 1.2 and  3.; 

-  4 

(g)  officially  brucellosis-free Region : 

means  a  part  of  a  Member  State  which  satisfies  the  conditions  laid 

down  in Annex  A  II 7.8  and  9.; 

(h)  officially brucellosis-free Member  State : 

means a Member  State which satisfies the conditions  laid down  in Annex 

A  II 10.11  and 12.; 

(i)  brucellosis-free bovine holding : 

means  a  bovine  holding  which  satisfies  the  conditions  laid  down  in 

Annex  A  11 4.5  and  6; 

(J)  Enzootic bovine  leucosis free holding : 

means  a  holding  which  satisfies  the  conditions  laid  down  in Annex  D. 

Chapter  1. Sections A and B. 

(k)  enzootic bovine  free Member  State or  region : 

means  a  region or  Member  State which meets  the  requirements  laid down 

in Annex  D. Chapter  I sections E.F  and G. 

(I)  official  veterinarian : 

means  the  veterinarian  designated  by  the  competent  central  authority 

of  the Member  State; 

Art icle 3 

1. Each Member  State shall  ensure that only animals that  fulfil  the conditions 

laid  down  in this Directive, where applicab le. are sent  from  its territory 

to that of another Member State. 

2. Bovine animals and swine covered by  this directive must  : 

(a)  be subjected  to a health examination and  identity check by an official 

veterinarian and show no clinical  sign of disease; 

-  5  -

(b)  not  have  been  obtained  from  a  holding  or  an  area  which  for  health 

reasons  is  subject  to  prohibition  affecting  the  species  involved  in 

accordance  with  Community  or  National  legislation. 

(c)  be  identified  as  provided  for  in  Council  Directive  92/102/EEC; 

(d)  not  be  animals  which  are  to  be  slaughtered  under  a  contagious  or 

infectious disease  eradication  programme  of  a Member  State. 

(e)  comply  with  the  provisions  of  Article  4 and  Article  5. 

Art icle  4 

1. Bovine  animals  and  swine  covered  by  this  directive  must  not,  at  any  time 

from  leaving  the  holding  of  origin  to arrival  at  the  destination  in another 

Member  State,  come  in contact  with  cloven  hoofed  animals  other  than  animals 

that  fulfil  the  conditions  for  intracommunity  trade. 

2. Bovine  animals  and  swine  covered  by  this  directive  must  be  transported  in 

means  of  transport  which  have  first  been  cleansed  and  disinfected  with  a 

disinfectant  officially  authorized  in  the  Member  State  of  Origin.  The 

transport  vehicles  must  be  so  arranged  that  the  animals'  faeces,  litter  or 

fodder  cannot  flow or  fall  out  of  the  vehicle  during  transportât!c:. 

3. Rules  for  the  approval  of  sites  where  disinfection  may  be  carried  out  and 

the  procedures  necessary  to  ensure  and  verify  compliance  with  the 

requirements  of  paragraph  3  above  shall  be  determined  in  accordance  with 

the  provisions  of  Article  12. 

-  6  -

Art icle  5 

1• Bovine  animals  and  swine  covered  by  this  directive  must  be  accompanied 

during  transportation  to  the  destination  by  a  health  certificate  conforming 

to  Annex  F.  The  certificate  shall  consist  of  a  single  sheet  and  shall 

contain  a  serial  number.  It  shall  be  drawn  up  on  the  day  of  the  health 

examination,  in one  of  the official  languages of  the  country  of  destination 

at  least.  The  certificate  shall  be  valid  for  10  days  from  the  date  of  the 

health  examination.  However, where  the  health  examination  takes  place  after 

leaving  the  holding  of  origin,  as  provided  for  in  paragraph  2  below,  the 

certificate  shall  be  valid  for  10 days  after  leaving  the  holding  of  origin. 

2.  The  health  examination  and  the  issuing  of  the  health  certificate  for  a 

consignment  of  animals  may  be  carried  out  in  the  holding  of  origin  or  at 

the  point  of  loading.  In  the  latter  case,  the  certifying  official 

veterinarian  must  have  been  provided  with  the  evidence  issued  by  the 

official  veterinarian  for  the  holding  of  origin  either  comprising  an 

official  document  containing  the  necessary  information  or  in  the  form  of 

the  certificate  contained  in Annex  F  with  Sections  A  and  B  duly  completed 

and  certified.  The  address  of  any  interim  location  such  as  an  approved 

market  or  approved  assembly  point  (part  C)  must  also  be  completed  by  the 

official  veterinarian  responsible  for  the  holding  of  origin. 

3. The  official  veterinarian  responsible  for  the  interim  location  shall 

certify  arrival  of  the  animals  there  and  where  applicable  complete  the 

final  intracommunity  certification  (Annex  F.  Part  D ). Where  animals  do  not 

enter  an  interim  location  the  final  intracommunity  certification  shall  be 

completed  by  the  official  veterinarian  responsible  for  the  holding  of 

origin. 

4-  The  official  veterinarian  completing  Annex  F.  part  D  shall  be  responsible 

for  registering  the movement  on  the AN IMP  system. 

5.  Interim  locations used  in  intracommunity  trade  such  as  markets  or  assembly 

centres  must : 

- 7 -

a.  be  supervised  by  an  official  veterinarian  who  shall  ensure  that,  in 

particular,  the  provisions  of  Article  4.1  and  Article  4.2  are  complied 

with  and  who  shall  provide  the  necessary  certification  referred  to 

above.  The  official  veterinarian's  duties  may  be  further  specified  by  a 

decision  taken  under  the  procedure  in Article  12 

b.  be  located  in an  area  which  is not  subject  to  prohibition  in  accordance 

with  Community  legislation 

c  be  cleaned  before  use  and  disinfected,  as  required  by  the  official 

veterinarian  responsible  for  the  premises. 

d.  be  designated  as  approved  markets  and  assembly  centres  by  the  Member 

State  of  origin.  Such  approval  may  be  limited  to  a  particular  species  or 

to  animals  for  breeding  and  production  or  to  animals  for  slaughter.  The 

Member  State  shall  notify  the  competent  central  authorities  of  the  other 

Member  states  and  the  Commission  as  to which  markets  or  assembly  centres 

are  approved. 

6.  The  interim  location  may  also  be  an  officially  approved  market  located  in a 

Member  State  which  is  not  the  Member  State  of  destination,  in  this  case, 

the  certificate  in  Annex  F  (including  section  D)  must  be  completed  by  the 

official  veterinarian  responsible  in  the  Member  State  where  the  animals 

originate.  The  official  veterinarian  responsible  for  the  market  shall 

provide  certification  to  the  animals  destination  by  completing  a  second 

certificate,  as  in  Annex  F.  endorsing  it  with  the  serial  number  of  the 

original  and  attaching  it  to  the  original  or  to  a  copy  of  the  original 

cert ifIcate. 

-  8  -

Article  6 

1. Animals  for  breeding  or  production  must,  in addition  to  the  requirements  in 

Art icles  3.4  and  5: 

-  have  remained  in  the  holding  of  origin  for  a  period  of  30  days  prior  to 

loading  or  since  birth  where  the  animals  are  aged  less  than  30  days.  The 

official  veterinarian  must,  on  the  basis  of  the  official  identification 

provided  for  in Article  3.2c  and  official  records  be  satisfied  that  the 

animals  have  complied  with  this  condition  and  furthermore  that  the 

animals  have  originated  in  the  Community  or  been  imported  from  a  Third 

Country  in compliance  with  Community  animal  health  legislation. 

-  Animals  imported  from  a  third  country  into  a  Member  State  which  is  not 

that  of  ultimate  destination, must  be  transported  to  the Member  State  of 

destination  as  quickly  as  practicable  under  cover  of  certification 

issued  under  Article  7  of  Council  Directive  91/496/EEC.  Upon  arrival  at 

the  destination,  such  animals  must,  as  regards  any  further  movements 

submit  to  the  requirements  of  this  directive  and  in  particular  to  the 

residency  requirement  in  the  first  indent. 

2.  Bovine  animals  for  breeding  and  production  must,  in  addition  to  the 

requirements  in Articles  3.  4  and  5: 

(a)  come  from  an  officially  tuberculosis-free  bovine  holding  and  in  the 

case  of  animals  more  than  six  weeks  old,  have  reacted  negatively  to  an 

intradermal  tuberculin  test  carried  out  during  the  30  days  prior  to 

leaving  the  herd  of  origin,  in accordance  with  the  provisions  of  Annex 

B  point  32.d: 

This  intradermal  tuberculin  test  is  not  required  if  the  animals 

originate  in  a  Member  State  or  part  of  a  Member  State  recognised  as 

Officially  Tuberculosis  Free. 

- 9 -

(b)  in  the  case  of  non  castrated  animais  come  from  an  officially 

brucellosis-free  bovine  holding  and  if  more  than  12  months  old,  have 

shown  a  brucella  count 

lower  than  30  international  units  of 

agglutination  per  millilitre  when  given  a  sero-aggiutination  test 

carried  out  during  the  30 days  prior  to  leaving  the  herd  of  origin  and 

complying  with  the  provisions of  Annex  C  paragraph  A; 

This  seroagglutination  test  is  not  required  if  the  animais  originate 

in  a  Member  State  or  part  of  a  Member  State  recognised  as  Officially 

Brucellosis  Free. 

(c)  come  from  an  enzootic  bovine  leucosis  free  holding  within  the  meaning 

of  Article  2 (g); 

(d)  not,  at  any  time  from  leaving  the  holding  of  origin  to  arrival  at  the 

destination,  come  in  contact  with  animals  which  only  meet  the 

requirements  in Article  6.3  below. 

3. Bovine  animals  for  slaughter  must,  in  addition  to  the  requirements  in 

Articles  3.4  and  5  come  from  herds  that  are  officially  tuberculosis  free, 

enzootic  bovine  leucosis  free  and  in  the  case  of  uncastrated  bovines,  from 

herds  that  are officially  brucellosis  free. 

Art icle  7 

Animals  for  slaughter  which  have  been  taken  on  arrival  in  the  country  of 

destination  to a slaughterhouse must  be slaughtered  there  as soon  as  possible, 

in accordance  with  animal  health  requirements. 

Animals  for  slaughter  which  have  been  taken  on  arrival  in  the  Member  State  of 

destination  to  a  market,  under  whose  rules  all  animals  must  be  removed  after 

the  market  to  a  slaughterhouse  approved  for  this  purpose  by  the  competent 

central  authority,  must  be  slaughtered  at  that  slaughterhouse  not  later  than 

five  days  after  arriving  at  the  market. 

-  10 -

Art icle 8 

Any  person  or  persons  suspecting  the  presence  of  one  of  the  diseases  listed  in 

Annex  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, 

- 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, 

-  12 -

-  the 

results 

of 

surveillance 

testing 

based 

on 

serological, 

microbiological,  pathological  or  epidemiological  investigation  and  on 

the  fact  that  the  disease  must  by  law  be  notified  to  the  competent 

authorities, 

-  the  period  over  which  the  surveillance was  carried  out, 

-  where  applicable,  the  period  during  which  vaccination  against  the 

disease  has  been  prohibited  and  the  geographical  area  concerned  by  the 

prohibition, 

-  the  arrangements  for  verifying  the  absence  of  the  disease. 

2. The  Commission  shall  examine  documentation  submitted  by  Member  States.  The 

additional  guarantees, general  or  specific, which  may  be  required  in  intra-

Community  trade  may  be  defined  in  accordance  with  the  procedure  laid  down 

in Article  12. Such  guarantees  must  not  exceed  those which  the Member  State 

implements  nationally. 

3. The Member  State  concerned  shall  notify  the Commission  of  any  change  in the 

details  specified  in paragraph  1 which  relate  to  the  disease,  in  particular 

regarding  any  new  outbreaks  of  the  disease.  The  guarantees  defined  as  laid 

down  in paragraph  2  may,  in  the  light  of  such  notification,  be  amended  or 

withdrawn  in accordance  with  the  procedure  laid  down  in Article  12. 

Article  11 

The  Annexes  to  this Directive may  be modified  in accordance  with  the  procedure 

detailed  in Article  12 

In  derogation  to  the  provisions  of  this  Directive,  specific  conditions  for 

trade  in  animals  for  special  purposes,  and  in  particular  for  show  purposes, 

may  be  determined  in accordance  with  the procedure  in Article  12. 

-  13 

Article  12 

1. Where  the  procedure  laid  down  in  this  Article  is  to  be  used,  matters  shall 

without  delay  be  referred  by  the  chairman,  either  on  his  own  initiative  or 

at  the  request  of  a  Member  State,  to  the  standing  veterinary  committee 

(hereinafter  called  the  "committee  ") set  up  by  the  Council  Decision  of  15 

October  1968. 

2. Within  the  committee  the  votes  of  Member  States  shall  be  weighted  as 

provided  in Article  148(2) of  the  treaty.  The  chairman  shall  not  vote. 

3. The  representative  of  the  Commission  shall  submit  a  draft  of  the  measures 

to  be  adopted.  The  committee  shall  deliver  its  opinion  on  such  measures 

within  a  time  limit  set  by  the  chairman  according  to  the  urgency  of  the 

matters  concerned.  Opinions  shall  be  delivered  by  a majority  of  54  votes. 

4.  The  Commission  shall  adopt  the  measures  and  shall  apply  them  immediately 

where  they  are  in accordance  with  the  opinion  of  the  committee.  Where  they 

are  not  in accordance  with  the  opinion  of  the  committee  or  if no opinion  is 

delivered,  the  Commission  shall  without  delay  propose  to  the  Council  the 

measures  to  be  adopted. 

The  Council  shall  adopt  the measures  by  a qualified  majority. 

If,  within  three  months  from  the  date  on  which  the  proposal  was  submitted  to 

it,  the  Council  has  not  adopted  any  measures,  the  Commission  shall  adopt  the 

proposed  measures  and  apply  them  immediately. 

Article  13 

1. Where  the  procedure  laid  down  in  this  Article  is  to  be  used,  matters  shall 

without  delay  be  referred  by  the  chairman,  either  on  his  own  initiative  or 

at  the  request  of  a  Member  State,  to  the  standing  veterinary  committee 

(hereinafter  called  the  " committee  ")  set  up  by  the  Council  decision  of 

15 October  1968. 

-  14 -

2. Within  the  committee  the  votes  of  the  Member  States  shall  be  weighted  as 

provided  in Article  148  (2) of  the  treaty.  The  chairman  shall  not  vote. 

3. The  representative  of  the  Commission  shall  submit  a  draft  of  the  measures 

to  be  adopted.  The  committee  shall  deliver  its  opinion  on  such  measures 

within  two days. Opinions  shall  be delivered  by  a majority  of  54 votes. 

4. The  Commission  shall  adopt  the  measures  and  shall  apply  them  immediately 

where  they  are  in accordance  with  the opinion  of  the  committee.  Where  they 

are  not  in accordance  with  the opinion  of  the  committee  or  if no opinion  is 

delivered,  the  Commission  shall  without  delay  propose  to  the  Council  the 

measures  to be  adopted. 

The  Council  shall  adopt  the  measures  by  a  qualified  majority.  If  within  15 

days  from  the  date  on  which  the  proposal  was  submitted  to  it,  the  Council  has 

not  adopted  any  measures,  the Commission  shall  adopt  the  proposed  measures  and 

apply  them  immediately. 

Article  14 

The  rules  laid  down  in Council  Directive 90/425/EEC  of  26  June  1990  concerning 

veterinary  and  zootechnical  checks  in  intra-Community  trade  in  certain  live 

animals  and  products  with  a  view  to  the  completion  of  the  internal  market,  as 
last  amended  by  Council  Directive  92/60/EEC  of  30  June  199210  shall  apply  in 

particular  to  checks  at  origin,  to  the organization  of,  and  follow-up  to,  the 

checks  to  be  carried  out  by  the  country  of  destination,  and  to  the  safeguard 

measures  to be  implemented. 

Article  15 

This directive  is addressed  to  the Member  States. 

10 OJ  No L 268  24.09.92  p  75 

ANNEX A 

I. OFFICIALLY TUBERCULOSIS-FREE BOVINE  HOLDING 

1. A bovine holding  is officially  tuberculosis-free  if : 

(a)  all  the animals are free  from cli.iical  signs of  tuberculosis; 

(b)  all  the animals over  six weeks old  have  reacted  negatively  to at  least 

two  official  intradermal  tuberculin  tests  carried  out  in  accordance 

with  Annex  B.  the  first  six  months  after  the  elimination  of  any 

infection  from  the holding  and  the second  six months  later  or 

in the case where  the herd  has been assembled  solely  from animals  that 

originate  in Officially  Tuberculosis  Free  herds  the  first  test  shall 

be  carried  out  at  least  60  days  after  assembly  and  the  second  shall 

not be  required 

(c)  following  the completion  of  the  first  test mentioned  in  (b) above, no 

bovine animal  aged over  six weeks has been  introduced  into the holding 

unless  it  has  reacted  negatively  to  an  intradermal  tuberculin  test 

performed  and  assessed  according  to Annex  B  and  carried  out  either  in 

the  30  days  prior  to.  or  the  30  days  after  the  date  of  it's 

introduction  into the holding. 

This test  shall  not be necessary  in Member  States where  the  percentage 

of  bovine  holdings  infected  with  tuberculosis  is  less  than  0.2%  and 

where  the  animal  originates  in  an  Officially  Tuberculosis  Free 

holding. 

2. A bovine holding will  retain Officially  Tuberculosis  Free status  if; 

(a)  the conditions detailed  in 1.(a) and  1.(c) continue  to apply 

-  <6 

(b)  all  animals  entering  the  holding  come  from  holdings  of  the  Officially 

Tuberculosis  Free  status. 

(c)  all  animals  in  the  holding,  with  the  exception  of  calves  aged  under 

six  weeks  which  were  born  in  the  holding,  are  subjected  to  routine 

tuberculin  testing  in accordance  with  Annex  B at  yearly  intervals. 

However,  the  Commission,  in  accordance  with  the  procedure  in Article 

12.  may,  for  a  Member  State  or  part  of  a  Member  State  where  all  the 

bovine  holdings  are  subject  to  official  operations  to  combat 

tuberculosis, alter  the  frequency  of  the  routine  tests as  follows; 

if the percentage  of  bovine  holdings  infected  with  tuberculosis  is not 

more  than  1% on  average  during  the  two most  recent  annual  supervisory 

periods,  the  interval  between  routine  holding  tests  may  be  increased 

to two years; 

if the percentage  of  infected  bovine  holdings  is not  more  than  0.2% on 

average  during  the  two most  recent  biennial  supervisory  periods,  the 

interval  between  routine  tests may  be  increased  to three  years; 

if the percentage  of  infected  bovine  holdings  is not  more  than  0.1% on 

average  during  the  two most  recent  triennial  supervisory  periods,  the 

interval  between  routine  tests  may  be  increased  to  four  years  and/or 

the  age  at  which  animals  have  to undergo  these  tests may  be  increased 

to 24 months. 

The Commission  may  also  in accordance  with  Article  13. take a  decision 

increasing  the  freguency  of  routine  tuberculin  testing  if the  level  of 

disease  appears  to have  increased. 

3. The Officially  Tuberculosis  free status of  a holding  shall  be suspended  if; 

(a)  the conditions detailed  in 2 above  have  not  been  complied  with. 

(b)  an animal  is deemed  to have  reacted  positively  to a  routine  tuberculin 

test,  or  a  case  of  tuberculosis  has  been  diagnosed  at  routine  post 

mortem  examination. 

- 

l-( 

In these  cases,  the status shall  remain  suspended  until  such  time as all 

the  remaining  animals  over  six  weeks  of  age  have  reacted  negatively  to 

at  least  two  official  intradermal  tuberculin  tests  in  accordance  with 

Annex  B. the  first  one carried  out  at  least  two months  after  elimination 

of  the animal  from  the holding  and  the second one  at  least  42 days after 

the first. 

However  where  the  routine  holding  test  detailed  in  2(c)  above  has  not 

been performed  on  time, the status of  the holding  shall  not  be  suspended 

provided  that  the  test  is carried  out  not  later  than  60  days  after  it 

was  originally  due,  and  provided  that  subsequent  testing  takes  place 

according  to the original  timetable. 

(c)  the  holding  contains  animals  of  unresolved  status  as  described  in 

Annex  B  point  32.  In this case  the status of  the  holding  shall  remain 

suspended  until  the animals status has been  clarified. 

4-  A  Member  State  or  part  of  a  Member  State  may  be  declared  Officially 

Tuberculosis  Free  according  to  the procedure  in Article  12  if  it meets  the 

following conditions; 

a.  the  percentage  of  infected  bovine  holdings  has not  been more  than  0.01% 

for  6  consecutive  years  and  at  least  99.9% of  the  holdings  have  been 

declared  Officially  Tuberculosis  Free  for  10 years 

b. an  identification  system  making  it  possible  to  identify  the  herds  of 

origin  and  transit  for each  bovine animal  is  in existence 

c. all  bovine  animals  slaughtered  must  be  subjected  to  a  post  mortem 

examination  by  an official  veterinarian. 

v  ai.  suspected  cases  of  tuberculosis  must  La  fully  investigated, 

including  tracing  and  checking  any  herds  of  origin  or  transit  and 

carrying  out  all  appropriate  laboratory  examinations.  While  such 

examinations  take  place  the  officially  tuberculosis  free  status  of  the 

herds  of  origin  or  transit  shall  be  suspended  until  clinical  or 

laboratory  examinations  or  tuberculin  tests  have  ruled  out  the  presence 

of  bovine  tuberculosis. 

^ 

5. The  Member  State  or  part  of  a  Member  State  will  retain  Officially 

Tuberculosis Free status  If: 

a. The conditions 4.a to 4.d  above continue to apply 

b. when  a  case  of  tuberculosis  is  confirmed,  the  officially  tuberculosis 

free status of  the holding of orioi.i and  transit  is withdrawn. 

c.  the  Officially  Tuberculosis  free  status  of  holdings  where  tuberculosis 

has been confirmed  remains withdrawn until; 

-  all  the  animals  that  have  been  deemed  to  be  infected  have  been 

slaughtered 

-  disinfection of  the premises and utensils has taken place 

-  all  the  remaining  bovine  animals  over  six  weeks  of  age  have  reacted 

negatively  to  at  least  two  official  intradermal  tests  in  accordance 

with  Annex  B.  the  first  at  least  six  months  after  the  removal  of  the 

Infected animals, the second six months after  the first. 

6.  If  there  is evidence  of  a  significant  change  in  the  situation  as  regards 

tuberculosis  in  a  Member  State  or  part  of  a  Member  State  which  has  been 

recognised  as  Officially  Tuberculosis  Free,  the  Commission  may  in 

accordance  with  Article  13  take  a  decision  suspending  or  revoking  the 

status  and  requiring  routine  tuberculin  tests  to  be  carried  out  in 

accordance with one of  the schedules  in 2.d above. 

'  *\ 

I I.  OFFICIALLY BRUCELLOSIS FREE AND BRUCELLOSIS FREE BOVINE  HOLDINGS 

For  the purposes of  this Annex  A.11  "bovine  animals" means  all  bovine  animals 

with  the exception of males castrated  before  the age of  four months. 

1. A bovine holding  Is Officially  Brucellosis Free  if; 

(a)  it  contains  no  bovine  animals  which  have  been  vaccinated  against 

brucellosis, except  females which  have been  vaccinated  at  least  three 

years previously; 

(b)  all  the  bovine  animals  have  been  free  from  clinical  signs  of 

brucellosis  for at  least six months; 

(c)  all  the  bovine  animals  over  12 months  old  have  been  subjected  to one 

of  the following  test  regimes with  negative  results  in accordance with 

Annex  C; 

( •) 

two seroagglutinat ion  tests  at  an  interval  of  more  than  three 

months and  less than  twelve months 

(ii) 

three ring  tests at  three monthly  intervals followed at  least 

six weeks  later by a seroagglutination test 

(iii) 

two  buffered  brucella  antigen  tests  at  an  interval  of  more 

than  three months and  less than twelve months 

( iv) 

two  micro-agglutination  tests  at  an  interval  of  more  than 

three months and  less than twelve months. 

2. A bovine holding will  retain Officially  Brucellosis Free status  if; 

(a)  one  of  the  following  test  regimes  is  carried  out  annually  with 

negative result  in accordance with Annex  C; 

( i )  three  ring  tests  carried  out  at  intervals  of  at  least  three 

months 

- cJtt-

( i i) 

t h r ee  m i lk  E L I S A 's  c a r r i ed  o ut at  i n t e r v a ls  of at  least  t h r ee 

months 

(iii) 

two  ring  tests  carried  out  at  an  interval  of  at  least  three 

months  followed  at  least  six  weeks  later  by  a serological  test 

(iv) 

two milk  ELISA's  carried  out  at  an  interval  of  at  least  three 

months  followed  at  least  six weeks  later  by  a serological  test 

(v) 

two  serological  tests  carried  out  at  an  interval  of  at  least 

three months and  not more  than six months. 

However,  the  Commission,  in  accordance  with  the  procedure  in  Article 

12.  may,  for  a  Member  State  or  part  of  a  Member  State  which  is  not 

Officially  Brucellosis  Free  but  where  all  the  bovine  holdings  are 

subject  to  official  operations  to  combat  brucellosis,  alter  the 

frequency  of  the  routine  tests as follows; 

-  where  not  more  than  1%  of  bovine  holdings  are  infected,  it  may  be 

sufficient  to  carry  out  each  year  two  ring  tests  or  two  milk  Elisa 

at  an  interval  of  at  least  three months, or one  serological  test. 

-  where  at  least  99.8  %  of  bovine  holdings  have  been  recognized  as 

Officially  Brucellosis  Free  for  at  least  four  years,  the  interval 

between  checks  may  be  extended  to  two  years  and  the  checks  must  be 

carried  out  using  one  of  the  serological  tests  referred  to  in 

paragraph  7.(a)  below. 

(b)  all  bovine  animals  entering  the  holding  come  from  holdings  of 

Officially  Brucellosis  Free  status,  and  in the  case of  bovine  animals 

over  12 months old,  have  shown  a brucella  count  of  less  than  30  iu of 

agglutination  per  ml  when  given  a  sero-agglutination  test  in 

accordance  with  Annex  C  during  the  30 days  prior  to  introduction  into 

the  holding: 

However  the  sero-agglutination  test  described  above  need  not  be 

required  in  Member  States,  or  Regions  of  Member  States,  where  the 

percentage  of  bovine  holdings  infected  with  brucellosis  has  not 

exceeded  0.2  %  for  at  least  two years and where  the animal  comes  from 

an  official  brucellosis-free  bovine  holding  within  that  Member  State 

or  Region  and  has  not  during  transportai ion  come  into  contact  with 

bovine animals of  lesser status. 

(c)  Notwithstanding  2b  above,  bovine  animals  from  a  Brucellosis  Free 

bovine  holding  may  be  introduced  into an  Officially  Brucellosis  Free 

holding  if they  are  at  least  18 months old  and  if vaccinated  against 

brucellosis,  the  vaccination  was  carried  out  more  than  a  year 

previously. 

Such  animals must  have  shown  in the  30 days  prior  to  introduction, a 

brucella count  lower  than 30  iu of agglutination per ml  and a negative 

result  when given  a complement  fixation  test, both  in accordance with 

Annex  C. 

If.  however,  a  bovine  animal  from  a  Brucellosis  Free  holding  is 

introduced  into  an  Officially  Brucellosis  Free  bovine  holding, under 

these  provisions,  that  holding  shall  be  considered  to  be  Brucellosis 

Free for two years from the date on which  the animal  was  introduced. 

3. The  Officially  Brucellosis  free  status  of  a  holding  may  be  suspended  or 

withdrawn  if: 

(a)  the  conditions  detailed  in  paragraphs  1  and  2  above  have  not  been 

complied with or 

(b)  as  a  result  of  laboratory  tests  or  on  clinical  grounds  one  or  more 

bovine animals are suspected of having  brucellosis 

If one or more bovine animals  in an Officially Brucellosis Free holding are 

suspected  of  having  brucellosis,  the  status  of  the  holding  may  be 

suspended, rather  than withdrawn,  if the animal  or  animals  are  immediately 

destroyed or  isolated. 

Jt'ï 

Where  the  animal  has  been  destroyed,  the  suspension  may  be  lifted  if  two 

sero-agglutination  tests,  carried  out  in  accordance  with  Annex  C  on  all 

bovine  animals  in the  holding  over  12 months old,  show  a  count  lower  than 

30  iu of agglutination per ml. The first  test  shall  be carried out  at  least 

30  days  after  the  removal  of  the  animal  and  the  second  at  least  60  days 

later. 

Where  the animal  has been  isolated,  it may be reintroduced  into the holding 

and  the  status of  the  holding  may  be  restored,  if  it subsequently  shows a 

sero-agglut inat ion  count  lower  than  30  iu of  agglutination  per  ml  and  has 

given  a  negative  result  to  a  complement  fixation  test,  these  tests  being 

carried out  in accordance with Annex  C. 

Where,  as  a  result  of  laboratory  tests  or  epidemiological  investigations, 

brucella  infection  has  been  confirmed  in  a  holding,  the  status  of  that 

holding  shall  not  be  restored  until  all  bovine  animals  that  were  pregnant 

at  the time of  the outbreak  have given negative results to the above  tests, 

the final  test having being carried out  at  least 21 days after  calving. 

4. A bovine  holding  is Brucellosis Free  if  it complies with  the conditions  in 

1a. 1b. and  1c. except  that: 

(i)  female bovine animals may be vaccinated: 

-  before  the  age  of  six  months  old  with  live  strain  19  vaccine  or 

other  vaccines approved  under  the procedure  laid down  in Article  12 

Pi, 

-  before  the  age of  15 months  old  with  killed  45/20  adjuvant  vaccine 

which  has been officially  inspected and  recognised: 

(i i) bovine  animals  aged  under  30  months  which  have  been  vaccinated  with 

live  strain  19  vaccine  may  give  a  serum  agglutination  test  result 

greater  than  30  i.u.  but  less  than  80  i.u.  of  agglutination  per 

millilitre provided  that, on the complement  fixation  test, they give a 

result  less  than  30  EEC units  in  the  case of  females  vaccinated  less 

than  12  months  previously  or  less  than  20  EEC  units  in  all  other 

cases. 

l^ 

(iii)  in  addition  to  those  tests  listed  in  1(c).  the  following  test 

regimes  will  also  be  approved  in order  to  attain  Brucellosis  Free 

status; 

a. 

two buffered brucella antigen tests carried out  at an  interval of 

more than three months and  less than  12 months 

b. 

two micro-agglutination  tests carried  out  at  an  interval  of  more 

than three months and  less than  12 months 

in accordance with  the provisions of Annex  C. 

5. A bovine holding will  retain Brucellosis Free status  if: 

(i)  it  is subject  to one of  the testing  regimes  listed  in 2(a) above 

(i i) bovine  animals  entering  the  holding  comply  with  the  reouirements  of 

2(b) above or 

-  come  from  holdings  of  Brucellosis  Free  status,  and  in  the  case  of 

bovine animals over  12 months old, have shown,  in the 30 days prior 

to  introduction  Into  the  holding,  less  than  30  iu of  agglutination 

per  ml  when  given  a  sero-agglut inat ion  test  and  a  negative 

complement  fixation test  in accordance with Annex C or. 

-  come  from  holdings  of  Brucellosis  Free  status,  be  aged  under  30 

months and  have been vaccinated with  live strain  19 vaccine may give 

a serum agglutination test  result greater  than 30  i.u. but  less than 

80  i.u.  of  agglutination  per  millilitre  provided  that,  on  the 

complement  fixation  test, they  give a  result  less than  30 EEC units 

in the case of  females vaccinated  les s than  12 months previously or 

less than 20 EEC units  in ail other  cases. 

— o^Ly 

6. The  Brucellosis  free  status  of  a  holding  shall  be  suspended  or  withdrawn 

il: 

(a)  the  conditions  detailed  in paragraph  4 and  paragraph  5 above  have  not 

been complied with or 

(b)  as  a  result  of  laboratory  tests  or  on  clinical  grounds  one  or  more 

bovine animals aged over  30 months are suspected  of  having  brucellosis 

If one  or  more  bovine  animals, aged  over  30 months,  in an Brucellosis  Free 

holding  are  suspected  of  having  brucellosis, the  status of  the  holding  may 

be  suspended,  rather  than  withdrawn,  if  the  animal  or  animais  are 

immediately  destroyed  or  isolated. 

Where  the  animal  has  been  destroyed,  the  suspension  may  be  lifted  if  two 

sero-agglutination  tests,  carried  out  in  accordance  with  Annex  C  on  all 

bovine  animals  in  the  holding  over  12 months  old,  show  a  count  lower  than 

30  iu of  agglutination per ml. The  first  test  shall  be carried  out  at  least 

30  days  after  the  removal  of  the  animal  and  the  second  at  least  60  days 

later. 

Where  the animal  has been  isolated,  it may be  reintroduced  into the  holding 

and  the  status  of  the  holding  may  be  restored,  if  it subseguently  shows  a 

sero-agglut inat ion  count  lower  than  30  iu of  agglutination  per  ml  and  has 

given  a  negative  result  to  a  complement  fixation  test,  these  tests  being 

carried out  in accordance with Annex  C. 

Where,  as  a  result  of  laboratory  tests  or  epidemiological  Investigations, 

brucella  infection  has  been  confirmed  in  a  holding,  the  status  of  that 

holding  shall  not  be  restored  until  all  bovine  animals  that  were  pregnant 

at  the  time of  the outbreak  have given negative results  to the above  tests, 

the final  test  having  being  carried  out  at  least  21 days after  calving. 

-2 s 

7. A  Region  of  a  Member  State  may  be  declared  Officially  Brucellosis  Free 

according  to  the  procedure  In  Article  12  If  it  meets  the  following 

conditions: 

a. no  case  of  abortion  due  to  brucella  infection  has  been  recorded  for  at 

least  three  years  and  at  least  99.8% of  the  holdings  have  been  declared 

Officially  Brucellosis  Free for  10  /ears 

b. an  identification  system  making  it  possible  to  identify  the  herds  of 

origin and  transit  for each  bovine animal  is  in existence 

8. Subject  to  point  9  below,  a  region  declared  Officially  Brucellosis  Free 

shall  retain  this  status  if  all  bovine  animals  over  24  months  old  are 

subjected  to  either  two  ring  tests  or  one  serological  test  every  three 

years.  In  the  event  of  a  positive  result  the  provisions  of  point  6  above 

shall  apply. 

9. A  region  declared  Officially  Brucellosis  Free  shall  report  the  occurrence 

of all  cases of Brucellosis to the Commission. The Commission may  according 

to  the  procedure  in  Article  13  propose  that  the  status  be  suspended  or 

revoked  and  recuire  that  routine  brucellosis  testing  be  carried  out  in 

accordance with one of  the schedules  in paragraph  2 above. 

10. 

A Member  State may  be  declared  Officially  Brucellosis Free  according  to 

the procedure  In Article  12  if  it meets the following conditions; 

a.  no  case of  abortion  due  to brucella  infection  has been  recorded  for 

at  least  three  years  and  at  least  99.8% of  the  holdings  have  been 

declared  Officially  Brucellosis  Free  for  10 years 

b.  an  identification system making  it possible  to  identify  the herds of 

origin and  transit  for each bovine animal  is  in existence 

M 

11. 

A  Member  State  declared  Officially  Brucellosis  Free  shall  retain  this 

Status  if; 

-  every  bovine  animal  suspected  of  being  infected  with  brucellosis  is 

notified 

to 

the 

competent 

authority 

and 

undergoes 

official 

investigation  for  brucellosis  including  at  least  two  serological  blood 

tests 

including 

the  complemei.c 

fixation 

test 

as  well 

as  a 

microbiological  examination  of  appropriate  samples  taken  in  the  case 

of  an  abort ion 

-  during  the  period  of  suspicion  which  shall  continue  until  negative 

results  have  been  obtained  from  the  tests  provided  for  in  the  first 

indent,  the  officially  Brucellosis  Free  status  of  the  herd  of  origin 

or  transit  of  the  suspected  bovine  shall  be  suspended. 

-  in  the  event  of  a  positive  result,  the  provisions  of  point  6  above 

apply. 

12. 

A  Member  State  declared  Officially  Brucellosis  Free  shall  report  the 

occurrence  of  all  cases of  Brucellosis  to  the Commission.  The  Commission 

may,  according  to  the  procedure  in Article  13 propose  that  the  status  be 

suspended  or  revoked  and  require  that  routine  brucellosis  testing  be 

carried  out  in  accordance  with  one  of  the  schedules  in  paragraph  2 

above. 

13.(a)  For  the  purposes  of  this  Annex  A . M.  a  serological  test  shall  mean 

either  a  serum  agglutination  test,  buffered  brucella  antigen  test, 

complement  fixation  test,  plasma  agglutination  test,  plasma  ring  test, 

micro  agglutination  test  or  individual  blood  ELISA.  as  described  in 

Annex  C. 

(b)  Where  ring  tests  are  carried  out  on  bulk  tanks,  the  number  of  those 

tests  referred  to  in  this  Annex  shall  be  doubled  and  the  intervals 

between  the  tests  shall  be  halved. 

ANNEX  B 

(Standards  for  the manufacture  and use of  bovine  and  avian  tuberculins) 

1. Officially  supervised  tuberculin  tests must  be carried  out  with  PPD or  HCSM 

tuberculins. 

2. Manufacturers'  working  standards  for  the  control  of  bovine  PPD  and  HCSM 

tuberculins  must  be  calibrated  in  community  tuberculin  units  (CTU) 

following biological  assay  against  the appropriate  EEC standard  tuberculin. 

3. Manufacturers' working  standards  for  the control  of  avian  tuberculins  must 

be calibrated  in  international  units  following  biological  assay  against  the 

EEC standard  for  PPD of  avian  tuberculin. 

4. The  EEC  standard  for  PPD  of  bovine  tuberculin  is  that  supplied  by  the 

Centraal  Diergeneeskundig  Instituut, Afdeling  Rotterdam,  The Netherlands. 

5. The  EEC  standard  for  bovine  HCSM  tuberculin  is  that  supplied  by  the 

Institut  Pasteur, Paris, France. 

6. The  EEC  standard  for  avian  tuberculin  is  that  supplied  by  the  Central 

Veterinary  Laboratory, Weybridge, Surrey,  England. 

7. Bovine  tuberculins  must  be  prepared  with  one  of  the  mycobacter ium  bovis 

strains  indicated  below : 

(a) AN5; 

(b) Vallée. 

8. Avian  tuberculins  must  be  prepared  with  one  of  the  mycobacter ium  avium 

strains  indicated  below  : 

(a) D4ER; 

(b) TB56. 

'« 

9. 

The ph of  tuberculins must  be between 6,5  and 7,5. 

10.  Antimicrobial  preservatives or  other  substances  that  may  be  added  to a 

tuberculin  shall  have  been  shown,  to  the  satisfaction  of  the  state 

institute responsible  for  the official  testing of  the tuberculin, not  to 

impair  the safety  and effectiveness of  the product. 

The  following  are  the  maximum  permitted  concentrations  for  phenol  and 

glycerol  : 

(a) phenol  :  0,5 % m/v; 

(b) glycerol  : 10 % v/v. 

11. 

Provided  the  tuberculins are  stored  at  a temperature  between  2 and  8'C, 

protected  from  light,  they  may  be  used  up  to  the  end  of  the  following 

periods subsequent  to the  last satisfactory potency  test  : 

(a)  liquid PPD tuberculins  : two years, 

lyophilized PPD tuberculins  :eight years-, 

(b) HCSM  tuberculins diluted  : two years. 

12. 

The  state  institutes  listed below  shall  be  responsible  for  the official 

testing of  tuberculins  in their  respective countries : 

(a) Germany  :  Paul-Ehrlich  institut,  Frankfurt/Main; 

(b) Belgium  :  Instituut  voor  hygiene  enepidemiologie,  J.Wytsmanstraat 

14, B-1050 Brussels; 

(c) France 

Laboratoire  national  des  medicaments  vétérinaires, 

Fougères; 

(d) Grand  Duchy of Luxembourg  : Institute of  the supplying  country; 

(e)  Italy: 

Istituto superiore di sanita, Rome; 

-=^V 

(f) Netherlands 

Centraal 

diergeneeskundig 

instituut,  afde ling 

Rotterdam; 

(g) Denmark 

Statens  Veterinaere  Serum laboratorium,  Copenhagen 

V; 

(h)  Ireland 

institute of  *he supplying  country; 

(i) United  Kingdom: 

Central  Veterinary  Laboratory, Weybridge,  Surrey. 

(J) Greece  : 

(k) Spain  :  Laboratorio de Sanidad  y produce ion Animal  de  Granada 

(I) Portugal  :  Laboratorio  Nacional  de  Investigacao  Veeterinaria  -

L i sboa 

13. 

Official  testing  must  be  carried  out  on  each  batch  of  bottled 

tuberculins  ready  for  use. 

14. 

Tuberculins shall  be  tested  by biological  and  chemical  methods. 

15. 

Tuberculins  must  be  sterile.  Tests  for  sterility  shall  be  carried  out 

according  to the specifications of  the european  pharmacopoeia. 

16. 

A  test  for  the  absence  of  toxic  or  irritant  properties  shall  be  carried 

out  according  to the specifications of  the european  pharmacopoeia. 

17. 

Tuberculins  must  be  chemically  analyzed  to  determine  the  concentration 

of  glycerol  and/or  phenol  and  also  the  concentration  of  any  other 

preservative which  may  have been  added. 

18. 

A  test  of  non-sensitization  to  tuberculin  must  be  carried  out  according 

to  the specifications of  the european  pharmacopoeia. 

V 

19. 

The  potency  of  tuberculins  must  be  assessed  by  biological  methods.  These 

methods  must  be used  for  HCSM  and  PPD  tuberculins-,  they  are  based  on  the 

comparison  with  standard  tuberculins of  the  tuberculins  to be  tested. 

20. 

The  protein  content  of  PPD  tuberculin  must  be  estimated  by  the  k je IdahI 

method.  The  nitrogen  is  converted  into  tuberculo-protein  content  by 

multiplying  by  a factor  of  6,25. 

21. 

The  EEC  standard  for  bovine  HCSM  has  a  potency  of  65  000  community 

tuberculin  units  (CTU) per  ml  and  is dispensed  in ampoules  containing  5 

ml  of  tuberculin. 

22. 

The  EEC  standard  for  bovine  PPD  has  a  potency  of  50  000  community 

tuberculin  units  (CTU)  per  mg  of  PPD  and  is  dispensed  lyoph i I i zed  in 

ampoules  containing  1,8  mg  of  PPD,  i.e.  0,00002  mg  PPD  has  a  potency 

equal  to one  community  tuberculin  unit. 

23. 

The  EEC  standard  for  avian  PPD  has  a  potency  of  50  000  international 

units  (i.u.)  per  mg  of  the  dried  material  of  the  purified  protein 

derivative  and  is  dispensed  in  the  lyophilized  in  ampoules  containing 

10mg of  PPD  plus  26,3 mg of  salts,  i.e. 0,0000726  mg  of  the standard  has 

a  potency  equal  to one  international  unit. 

24. 

Tuberculins  submitted  by  manufacturers  for  testing  by  the  state 

institutes  listed  in paragraph  12 must  have  been  tested  for  potency  by 

biological  assay  against  the  appropriate  standards  as  listed  in 

paragraphs  2 and  3. 

25.(a)  potency  testing on  guinea  pig 

Albino  guinea-pigs  weighing  between  400  and  600  g must  be  used.  These 

guinea-pigs  must  be  in  good  health  at  the  time  of  injection  of  the 

tuberculin.  Not  less  than  eight  guinea-pigs  shall  be  used  for  each 

assay.  The  assay  should  be  made  not  less  than  one  month  after 

sens it izat ion. 

•V\ 

(aa) for  the  assay  of  bovine  tuberculins,  guinea-pigs  shall  be  sensitised 

by  one  of  the  following  methods  : 

1. 

The  injection  of  heat-killed  mycobacterium  bovis  strain  an5  in 

oiI  adjuvant, 

2. 

The  injection  of  living  •Mycobacterium  bovis  strain  an5  in 

physiological  saline, 

3. 

The  injection of  beg  vaccine. 

(bb) for  the  assay  of  avian  tuberculins  guinea-pigs  shall  be  sensitized  by 

injection  of  2  mg  of  heat-killed  avian-type  tubercular  bacilli 

suspended  in 0,5  ml  of  sterile  liquid  paraffin  or  by  the  injection  of 

live avian-type  tubercular  bacilli  in physiological  saline. The  avian-

type  strain  d4 must  be used  for  this  purpose. 

(cc) each  tuberculin  under  test  shall  be  assayed  against  the  appropriate 

standard  tuberculin  by  an  intradermal  assay  using  groups  of  guinea-

pigs  suitably  sensitized. 

The  hair  shall  be  clipped  from  both  sides  of  each  guinea-pig.  The 

assay  shall  be  carried  out  by  comparing  the  reactions  induced  by  a 

series  of  intracutaneous  injections  of  doses  of  not  more  than  0,2  ml 

of  dilutions  of  the  standard  tuberculin  in  isotonic  buffered  saline 

solution  containing  Tween  80, 0,0005%,  with  a  corresponding  series  of 

injections  of  the  tuberculin  under  test.  Dilutions  shall  be  arranged 

in  geometric  series,  and  injected  into  guinea-pigs  according  to  a 

randomized  latin  square  design  (four  sites  on  each  side  of  an  eight-

point  assay  is  used).  The  diameters  of  the  reactions  at  each  site 

should  be measured  and  recorded  after  24  to 28  hours. 

For  each  sample  of  tuberculin  under  test,  an  estimate  of  relative 

potency  against  the  appropriate  standard  and  its  fiducial  limits shall 

be  made  by  statistical  methods,  using  the  diameters  of  the  reactions 

and  the  logarithms  of  the  doses  as  metameters.  The  bovine  tuberculin 

under  test  is  of  acceptable  potency  if  its  estimated  potency 

guarantees  per  bovine  dose  2000  community  tuberculin  units  (more  or 

less  25 %)  in cattle. The  potency  of  each  tuberculin  under  test  shall 

be  expressed  as  appropriate  in  community  tuberculin  units  or 

international  units  per ml. 

(b)  potency  testing on  cattle 

Periodic  potency  testing  of  bovine  tuberculins  may  be  carried  out  on 

naturally  or  artificially  infected  tuberculous  cattle.  These  potency 

tests,  on  groups  of  tuberculous  cattle,  shall  be  carried  out  by 

intradermal  four  or  six-point  assay  of  the  tuberculin  under  test 

against  the  appropriate  standard  and  the  potency  of  the  tuberculin 

shall  be estimated  by  statistical  methods  as  in the guinea-pig  assay. 

26. 

The  following  requirements  shall  apply  to  the  labelling  of  tuberculin 

containers  and  packages  : 

The  label  on  the containers  and  the  label  on  the package  shall  state : 

-  the name of  the  preparation, 

-  for  liquid  preparations,  the  total  volume  in the  container, 

-  the number  of  community  or  international  units per ml  or  per  mg, 

-  the manufacturer's  name, 

-  the batch  number, 

-  the  nature  and  quantity  of  the  reconstituting  liquid  for  the  freeze-

drled  préparât ion. 

The  label  on  the container  or  the  label  on  the package  shall  state: 

-  the expiry  date, 

-  the conditions of  storage, 

-  the name  and,  if possible,  the proportions of  any  added  substance, 

-  the strain of  bacillus  from which  the  tuberculin  has been made. 

27. 

Community  laboratories  designated  in  accordance  with  the  procedure  in 

Article  12  will  be  made  responsible  for  the  additional  examination  of 

routine  issue  field  tuberculins used  in the Member  States  to ensure  that 

the  potency  of  each  of  these  tuberculins  is adequate  in  relation  to  the 

appropriate  community  standard  tuberculin.  These  examinations  must  be 

carried  out,  in  tuberculous  bovines,  in suitably  sensitized  guinea-pig^ 

and  by  appropriate  chemical  tests. 

28. 

The  following  shall  be  recognized  as  official  intradermal  tuberculin 

tests: 

(a) the  single  intradermal  test  -  this  test  requires  a  single  injection 

of  bovine  tuberculin. 

(b) the  intradermal  comparative  tesc  -  this  test  requires  one  injection 

of  bovine  tuberculin  and  one  injection  of  avian  tuberculin  given 

simultaneously. 

29. 

The  dose of  tuberculin  injected  shall  be  : 

1. Not  less  than  2 000 CTU  of  bovine  tuberculin; 

2.  Not  less  than  2 000  iu of  avian  tuberculin.WI5 

The  volume of  each  injection  dose  shall  not  exceed  0,2  ml. 

30. 

Tuberculin  tests  shall  be  carried  out  by  injecting  tuberculin(s)  into 

the  skin  of  the  neck.  The  injection  sites  shall  be  situated  at  the 

border  of  the  anterior  and  middle  thirds  of  the  neck.  When  both  avian 

and  bovine  tuberculins  are  injected  in  the  same  animal,  the  site  for 

injection  of  avian  tuberculins  shall  be  about  10  cm  from  the  crest  of 

the  neck  and  the  site  for  the  injection  of  bovine  tuberculin  about  12,5 

cm  lower  on  a  line  roughly  parallel  with  the  line of  the  shoulder  or  on 

different  sides of  the  neck;  in young  animals  in which  there  is not  room 

to  separate  the  sites  sufficiently  on  one  side  of  the  neck,  one 

injection  shall  be  made  on  each  side  of  the  neck  at  identical  sites  in 

the  centre of  the middle  third  of  the  neck. 

31. 

The  technique  of  tuberculin  testing  and  interpretation  of  reactions 

shalI  be  as  fol lows : 

(a) technique  : 

Injection  sites  shall  be  clipped  and  cleansed.  A  fold  of  skin  within 

each  clipped  area  shall  be  taken  between  the  forefinger  and  thumb 

and  measured  with  calipers  and  recorded.  A  short  sterile  needle, 

bevel  edge  outwards,  with  graduated  syringe  charged  with  tuberculin 

attached  shall  be  inserted  obliquely  into  the  deeper  layers  of  the 

skin.  The  dose  of  tuberculin  shall  then  be  injected.  A  correct 

injection  shall  be  confirmed  by  palpating  a  small  peal ike  swelling 

at  each  site of  injection. The  skin-fold  thickness of  each  injection 

site  shall  be  remeasured  72  hours after  injection  and  recorded. 

(b)  interpretation of  reactions  : 

The  interpretation  of  reactions  shall  be  based  on  clinical 

observations and  the  recorded  increases(s)  in skin-fold  thickness at 

the sites of  injection  72 hours after  injection  of  tuberculin(s). 

(ba) 

negative  reaction  : if only  limited  swelling  is observed, with  an 

increase  of  not  more  than  2  mm  in  the  thickness  of  the  fold  of 

skin  without  clinical  signs  such  as  diffuse  or  extensive  oedema, 

exudation,  necrosis,  pain  or  inflammation  of  the  lymphatic  ducts 

in that  region or  of  the  lymph  nodes. 

(bb) 

inconclusive  reaction  : if no clinical  signs  such  as mentioned  in 

(ba)  are  observed  and  if  the  increase  in  skin-fold  thickness  is 

more  than  2 mm  and  less than  4mm. 

(be) 

positive  reaction  :  if  clinical  signs  such  as  mentioned  in  (ba) 

are  observed  or  there  is  an  increase  of  4  mm  or  more  in  the 

thickness of  the  fold of  skin  at  the  injection  site. 

32. 

The  interpretation  of  official  intradermal  tuberculin  tests  shall  be  as 

fo11ows : 

(a) single  intradermal  test  : 

positive  : 

a positive bovine  reaction  as defined  in paragraph  31 

(be); 

inconclusive  : an  inconclusive  reaction  as  defined  in  paragraph  31 

(bb); 

negative  : 

a  negative  bovine  reaction  as  defined  in  paragraph 

31(ba). 

Animals  inconclusive  to  the  single  intradermal  test  shall  be  subjected 

to  another  test  after  a  minimum  of  42  days. 

Animals  which  are  not  negative  to  this  second  test  shall  be  deemed  to 

be  positive  to  the  test. 

Animals  positive  to  the  single  intradermal  test  may  be  subjected  to  an 

intradermal  comparative  test; 

(b)  intradermal  comparative  test  for  the  establishment  and  maintenance 

of  officially  tuberculosis-free  holding  status  : 

pos i t i ve 

a  positive  bovine  reaction  which  is  more  than  4mm 

greater  than  the  avian  reaction,  or  the  presence  of 

cl inical  signs; 

inconclusive  : a  positive  or  inconclusive  bovine  reaction  which  is 

from  1  to  4  mm  greater  than  the  avian  reaction,  and 

the  absence  of  clinical  signs; 

negat ive 

a  negative  bovine 

reaction,  or 

a  positive  or 

inconclusive  bovine  reaction  but  which  is equal  to  or 

less  than  a  positive  or  inconclusive  avian  reaction 

and  the  absence  of  clinical  signs  in both  cases. 

Animals  inconclusive  to  the  intradermal  comparative  test  shall  be 

subjected  to  another  test  after  a  minimum  of  42  days.  Animals  which 

are  not  negative  to  this  second  test  shall  be  deemed  to  be  positive  to 

the  test; 

(c) Officially  tuberculosis-free  holding  status  may  be  suspended  and 

animals  from  the  holding  shall  not  be  allowed  to  enter  intra 

community  trade  until  such  time  as  the  status  of  the  following 

animals  is  resolved: 

1.  Animals  which  have  been  deemed  to  be  inconclusive  to  the  single 

intradermal  tuberculin  test; 

2. Animais  which  have  been  deemed  to  be  positive  to  the  single 

intradermal  tuberculin  test  but  are  awaiting  retest  with  an 

intradermal  comparative  test; 

3. Animals  which  have  been  deemed  to  be  inconclusive  to  the 

intradermal  comparative  test. 

(d) Where  animals  are  required  by  Community  legislation  to  be  subjected 

to  an  Intradermal  test  prior  to  movement,  the  test  shall  be 

interpreted  so  that  no  animal  which  shows  an  increase  in  skin  fold 

thickness  greater  than  2mm  or  the  presence  of  clinical  signs  is 

entered  into  intracommunity  trade. 

ANNEX  C 

BRUCELLOSIS 

A.  Serum  agglutination  tests 

1. The  standard  agglutinating  serum  must  conform  to  the  standard  serum 

prepared  by  the Veterinary  Laboratory,  Weybridge,  Surrey,  England. 

The  ampoule  must  contain  1  000  iu  of  agglutination  obtained  by 

lyophiIizing  1 ml  of  bovine  serum. 

2. The  standard  serum  must  be  that  supplied  by  the  Bundesgesundheitsamt, 

Berlin. 

3. The  degree  of  brucella  agglutination  in a  serum  must  be  expressed  in  iu 

per  ml  (i.e. Serum  x - 80  iu/ml). 

4. Readings  of  slow  sero-agglut inat ion  in  tubes  must  be  taken  at  50  or  at 

75%  agglutination,  the  antigen  used  having  been  titrated  under  identical 

conditions against  the  standard  serum. 

5. The  agglutinating  value  of  various  antigens  in  relation  to  standard 

serum  must  be within  the  following  limits  : 

-  if the  reading  is made  at  50 %  : between  1/600  and  1/1000, 

-  if the  reading  is made  at  75 %  : between  1/500  and  1/750. 

6. Weybridge  strain  no  99  and  USDA  1119  or  any  other  strain  of  equivalent 

sensitivity  must  be  used  for  preparing  the  antigen  for  use  in  tube 

agglutination  (slow  method). 

7.  The  culture  media  used  for  keeping  the  strain  in  the  laboratory  and  for 

producing  the  antigen  must  be  such  that  they  do  not  encourage  bacterial 

dissociation  (s minus  r ); potato  agar  should  preferably  be  used. 

8. The  bacterial  emulsion  must  be  made  from  physiological  saline  (NaCI 

8,5%)  phenolized  at  5 %.  Formol  must  not  be  used. 

9. The official  institutes  indicated  below must  be made  responsible  for  the 

official  testing  of  antigens  : 

(a) Germany: Bundesgesundheitsamt, uerlin; 

(b) Belgium:  Institut  national  de  recherches  vétérinaires, Brussels; 

(c) France: Laboratoire  central  de  recherches  vétérinaires, Alfort; 

(d) Grand  Duchy  of  Luxembourg  : Institute of  the supplying  country; 

(e)  Italy:  Istituto  superiore  di  sanita, Rome; 

(f) Netherlands  :  Centraal 

Diergeneeskundig 

Instituut, 

Afde ling, 

Rotterdam 

(g) Denmark: Statens Veterinaere  Serum laboratorium, Copenhagen  v.; 

(h)  Ireland:  Veterinary  Research  Laboratory,  Department  of  Agriculture 

and  Food, Dub lin; 

(i) United  Kingdom  : 

-  Great  Britain  : 

the 

Central 

Veterinary 

Laboratory, 

-  Northern  Ireland  : 

Veterinary 

Research 

Laboratory, 

Weybridge,Surrey,  England, 

(J) Greece 

Stormont.Belfast. 

(k) Spain  : Centro  Nacional  de brucelosis  de  Murcia 

(I) Portugal: Laboratoria  Nacional  de  Investigacao Veterinaria  -  Lisboa 

10.  Antigens  may  be  delivered  in  the  concentrated  state  provided  the 

dilution  factor  to be used  is  indicated  on  the bottle  label. 

11.  In  order  to  carry  out  a  sero-agglut inat ion  test,  at  least  three 

dilutions  must  be  prepared  for  each  serum.  Dilutions  of  suspect  serum 

must  be  made  in  such  a  way  that  the  reading  of  the  reaction  at  the 

infection  limit  is  made  in  the  median  tube.  If  there  is  a  positive 

reaction  in  this  tube,  the  suspect  serum  contains  at  least  30  iu of 

agglutination  per  ml. 

B. Complement  fixation  reaction  test 

1. The  standard  serum  is  the  same  as  that  under  A.1  of  this  Annex.  In 

addition  to  its  content  in  international  agglutinating  units,  1  ml  of 

this  lyophilized  bovine  serum  must  contain  1 000 sensitizing  units  which 

fix  the  complement.  These  sensitizing  units  are  called  EEC  sensitizing 

units. 

2. The  standard  serum  must  be  supplied  by  the bundesgesundheitsamt,  Berlin. 

3. A  serum's  level  of  antibodies which  fix  the complement  must  be  expressed 

in  EEC  sensitizing  units  (for  example  :  serum  x  -  60  EEC  sensitizing 

units per  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. 

8.  In  carrying  out  the  complement  fixation  reaction  test,  the  following 

controls must  be made each  time : 

(a) control  of  the anti-complementary  effect  of  the  serum; 

(b) control  of  the  antigen; 

(c) control  of  sensitized  red blood  corpuscles; 

(d) control  of  the  complement; 

(e) control  using  a  positive  serum  of  sensitivity  at  the  start  of  the 

react ion; 

(f) control  of  the specificity of  the  reaction using  a negative  serum. 

9. The  supervision  and  official  control  of  standard  serums  and  antigens 

shall  be carried  out  by  the bodies  listed  in a 9 of  this Annex. 

10.  Antigens  may  be  delivered  in  the  concentrated  state  provided  the 

dilution  factor  to be used  is  indicated on  the bottle  label. 

C. Ring  test 

1. The  ring  test  must  be made on  the  contents of  each  milk  churn  or  on  the 

contents of  each  bulk  tank  from  the  farm. 

2. The  standard  antigen  to  be  used  must  come  from  one  of  the  institutes 

listed  in paragraph  a.9  (a) to  (J). It  is recommended  that  the  antigens 

should  be standardized  according  to the WH0/FA0  recommendations. 

3. The  antigen  may  be  stained  only  with  haematoxylin  or  tetrazolium; 

haematoxylin should  preferably  be  used. 

4.  If  no  preservation  is used  then  the  reaction  test  must  be  carried  out 

between  18 and  24 hours of  taking  the sample  from  the cow.  If milk  is to 

be  tested  later  than  24  hours  after  sampling,  then  preservation  must  be 

used,  formalin or  mercuric  chloride  may  be used  as  preservatives  and  if 

either  of  these  are  used  the  test  must  be  carried  out  within  the 

following  14  days  after  the  day  of  sampling.  Formalin  may  be  added  to 

give  a  final  concentration  in  the  milk  sample  of  0,2  %  and,  in  such 

cases,  the  ratio  between  the  amount  of  milk  and  the  solution  of  formalin 

must  be  at  least  10  to  1.  A  solution  of  mercuric  chloride  may  be  used 

instead  of  formalin  to  give  a  final  concentration  in  the  milk  of  0,2  % 

and,  in  such  cases,  the  ratio  between  the  amount  of  milk  and  the 

solution  of mercuric  chloride  must  be  10 to  1. 

5. The  reaction  must  be carried  out  using  one of  the  following  methods  : 

-  on  a  column  of  milk  at  least  25 mm  high  and  on  a  volume  of  milk  of 1 

ml  to  which  0,03  ml  of  one  of  the  standardized  stained  antigens  has 

been  added, 

-  on  a  column  of  milk  at  least  25 mm  high  and  on  a  volume  of  milk  of 1 

ml  to  which  0,05  ml  of  one  of  the  standardized  stained  antigens  has 

been  added, 

-  on  a  volume  of  milk  of  8  ml  which  0,08  ml  of  one  of  the  standardized 

stained  antigens  has been  added, 

-  on  a  column  of  milk  at  least  25 mm  high  and  on  a  volume  of  milk  of  2 

ml  to  which  0,05  ml  of  one  of  the  standardized  stained  antigens  has 

been  added. 

6.  The  mixture  of  milk  and  antigens  must  be  incubated  at  37'C  for  not  less 

than  45 minutes  and  not  more  than  60 minutes. The  test  must  be  assessed 

within  15 minutes  of  removal  from  the  incubator. 

7. The  reaction  must  be  assessed  according  to  the  following  criteria  : 

(a) negative  reaction  :  coloured  milk, colourless  cream; 

(b) positive  reaction  :  milk 

and 

cream 

identically 

coloured 

or 

colourless milk  and  coloured  cream. 

D. The  buffered  brucella  antigen  test 

The  buffered  brucella  antigen  test  may  be  carried  out  using  one  of  the 

fol lowing methods  : 

a. Manual  test 

1. The  standard  serum  shall  be  the  second  international  standard  anti-

brucella  abortus  serum  which  is  supplied  by  the  Central  Veterinary 

Laboratory, Weybridge, Surrey,  England. 

2. The  antigen  shall  be  prepared  without 

reference  to  the  cell 

concentration,  but  its  sensitivity  must  be  standardized  in  relation  to 

the  second  international  standard  anti-brucella  abortus  serum  in such  a 

way  that  the antigen  produces a positive  reaction with  serum  dilution of 

1  : 47.5 and  a negative  reaction  with  a dilution of  1  : 55. 

3. The  antigen  shall  be suspended  in buffered  brucella  antigen  diluent  at a 

ph  of  3.65  more  or  less  0.5  and  may  have  been  stained  by  the  use  of 

rose bengal  dye. 

4. Weybridge  strain  no  99  or  usda  1119  or  any  other  strain  of  equivalent 

sensitivity must  be used  for  preparing  the  antigen. 

5. The  culture  media  used  for  keeping  the  strain  in  the  laboratory  and  for 

producing  the  antigen  must  be  such  that  they  do  not  encourage  bacterial 

dissociation  (s  -  r ); potato  agar  medium  or  continuous  culture  methods 

should  be  used. 

6.  The  antigen  shall  be  tested  against  eight  freeze-dried  known  positive 

and  negative sera. 

7. The official  supervision  and  control  of  standard  serum  and  antigen shall 

be carried  out  by  the official  bodies  listed  in Annex  C  (a) (9). 

8. The antigen  shall  be delivered  ready  for use. 

9. The buffered  brucella  antigen  test  shall  be carried  out  in the  following 

manner  : 

•q-h-

(a) one  drop  (0.03  ml)  of  antigen  should  be  placed  alongside  one  drop 

(0.03 ml) of  the  serum  on  a white  plate; 

(b) they  should  be  mixed  with  an  applicator  stick,  first  in  a  straight 

line and  then  in a circle of  about  10 to  12 mm  diameter; 

(c) the  plate  should  then  be  rocked  back  and  forth  for  four  minutes 

(about  30 times per  minute); 

(d) readings  should  be  taken  in a good  light;  if there  is no evidence of 

agglutination,  the  test  shall  be  regarded  as negative;  any  degree of 

agglutination  shall  be  regarded  as  positive,  unless  there  has  been 

excessive  drying  round  the edges. 

b. Automated  method 

The  automated  method  must  be  at  least  as  sensitive  and  accurate  as  the 

manual  method. 

E. Plasma  ring-test 

A. extraction  of  the  plasma 

The  tube  containing  blood,  coagulation  of  which  having  been  inhibited  by 

the  addition  of  edta,  should  be  centrifuged  for  three  at  3  000  r/min  and 

subsequently  kept  at  37"C  for  12 to 24 hours. 

B.  Evaluation 

0.2  ml  of  stabilized  plasma  should  be  placed  in  a  tube  with  1  ml  of 

untreated  milk.  After  mixing,  one  drop  (0.05  ml)  of  abr-antigen  should  be 

added  and  the  whole  again  mixed.  The  antigen  should  be  standardized  in 

relation  to  a standard  antigen  supplied  by  the  body  referred  to  in  (a)  (9) 

(a). 

Following  an  incubation  period  of  45  minutes  at  37'C,  a  reading  should  be 

taken  within  15  minutes.  The  result  shall  be  regarded  as  positive  if  the 

colour  of  the  ring  has become  the same  as, or  darker  than,  that  of  the milk 

coIumn. 

F.  Plasma  agglutination 

The  plasma  extracted  in accordance  with  e  (a) may  be used  immediately  after 

centrifuging,  no  thermal  stabilization  being  necessary.  0.05  ml  of  plasma 

should  be  mixed  with  1  ml  of  antigen  for  50  %  sero-agglut inat ion,  which 

corresponds  to  a  dilution  of  1  :  20  for  sero-agglutination.  A  reading 

should  be  taken  after  18  to  24  hours  incubation  at  37?C.  50%  or  more 

agglutination  shall  be  regarded  as  positive. 

G. Micro-agglutination  test 

1. Diluents  are  made  up  of  0,85  %  physiological  saline  solution  phenolized 

at  0,5  %. 

2. The  antigen  shall  be  prepared  as  described  under  points  6,  7  and  8  of 

Annex  C  (a) and  shall  be  titrated  as  described  under  point  5 of  Annex  C 

(a).  At  the moment  the antigen  is used  safranin  o  shall  be added  at  0,02 

%  (final  dilution). 

3. The  standard  serum  is the  same  as  that  under  point  1 of  Annex  C  (a). 4. 

The  standard  serum  must  be supplied  by  the bundesgesundheitsamt,  berlin. 

5. The  micro-agglutination  test  shall  be  carried  out  on  plates  bearing 

wells  with  conical  bottoms  of  a  volume  of  0,250  ml.  The  test  shall  be 

carried  out  as  follows : 

(a) predilution  of  the  serum  : 0,050 ml  of  each  serum  to  be  tested  are 

added  to each  well  containing  0,075 ml  of  diluent.  The  mixtures  are 

shaken  for  30 seconds. 

(b) gradual  serum  dilution  : prepare  at  least  three  dilutions  for  each 

serum,  to this end  from  the predilutions  (1  : 2,5) one  takes 0,025 

ml of each  serum and transfers  them to a well  containing  0,025 ml of 

diluent.  In this way the first  dilution  reaches a strength  of  1 : 5 

and the following dilutions are carried out by doubling. 

(c) addition  of  antigen  : 0,025  m<  of  antigen  is  added  to  each  well 

containing  the different  serum  dilutions. After  being  shaken  for 30 

seconds the plates are closed with  their  respective  lids and kept at 

37'C for 20 to 24 hours  in a humidified  atmosphere. 

(d) reading  the results  : assessment  of the aspect  of  the sedimentation 

of the antigen  is made by examining  the bottom of the well  reflected 

in  a  concave  mirror  placed  above  it.  If  there  is  a  negative 

reaction,  the  antigen  forms  a  sediment  in  the  form  of  a  compact 

button  with  clear  edges  and  having  an  intense  red colour.  If there 

is a positive  reaction, on  the other  hand, a diffused  pink  veil  is 

formed  that  is  evenly  distributed.  The  different  percentages  of 

agglutination  are  determined  by  comparison  with  antigen  checks 

indicating  0, 25, 50, 75 and 100 % agglutination. The title of each 

serum  is expressed  in  international  units of  agglutination  per ml. 

There  should  be  included  in  the  test,  controls  with  negative  and 

positive  serum  diluted  so as  to contain  30  international  units of 

agglutinât ion per ml. 

H. Enzyme-1 inked  immunosorbent  assay  (Eli sa) for detecting bovine brucellosis. 

1. The material  and reagents to be used are as follows: 

(a) solid  phase microplates, cuvettes or any other  solid phase; 

(b) the antigen  is fixed  to the solid  phase  with  or without  the aid of 

polyclonal or monoclonal  catching antibodies. 

(c) the biological  fluid  to be tested; 

(d) a corresponding positive and negative control; 

(e) conjugate; 

(f) a substrate  adapted  to  the enzyme  used; 

(g) a stopping  solution,  if  necessary; 

(h) solutions  for  the  dilution  of  che  test  samples  for  preparations  of 

the  reagents  and  for  washing; 

(i) a  reading  system  appropriate  to  the substrate  used. 

2. Standardization  and  sensitivity  of  test: 

1.  bulk  milk  samples  are  classified  negative  if  they  give  a  reaction 

less  than  50 % of  that  given  by  a  1  in 10 000 dilution  of  the  second 

international  brucellosis  standard  serum  made  up  in negative  milk; 

2.  individual  serum  samples  are  classified  negative  if  they  give  a 

reaction  less  than  10 %  of  that  given  by  a  1  in 200 dilution  of  the 

second  international  brucellosis  standard  serum  made  up  in  saline 

solution  or  in any other  recognized  dilution,  in accordance  with  the 

procedure  laid  down  in Article  12 after  receiving  the opinion  of  the 

Scientific  Veterinary  Committee. 

The  brucellosis  Elisa  standards  shall  be  as  specified  in Annex  C,  A. 1 

and A.2  (to be used  at  the dilutions  indicated  on  the  label). 

3. Conditions  for  use  of  the  Elisa  test  for  bovine  brucellosis 

The  Elisa  method  may  be  used  on  a  sample  of  milk  or  whey  taken  from  the 

milk  collected  from  a  farm  with  at  least  30 % of  dairy  cows  in milk. 

If  this  method  is  used,  measures  must  be  taken  to  ensure  that  the 

samples  taken  can  be  identified  with  the  animals  from  which  the milk  or 

sera  examined  were  taken. 

~  H r 

ANNEX  D 

CHAPTER I 

ENZOOTIC  BOVINE  LEUCOSIS  FREE  HOLDINGS. MEMBER  STATES  AND  REGIONS 

A. A  holding  is an  Enzootic Bovine  Leucosis  Free  holding  if: 

(i) 

there  is no evidence, either  clinical  or  as  a  result  of  a  laboratory 

test,  of  any  case  of  enzootic  bovine  leucosis  in  the  holding  and  no 

such  case  has been  confirmed  in the previous  two years, and 

(ii) 

all  animals over  24 months  of  age  have  reacted  negatively  during  the 

preceding  12 months  to  two  tests  carried  out  in accordance  with  this 

Annex,  at  an  interval  of  at  least  four  months, or 

( H i)  it  meets  the  requirements  of  (i)  above  and  is  situated  in  an 

enzootic  bovine  leucosis  free Member  State or  region. 

B. An  individual  holding  shall  retain  Enzootic Bovine  Leucosis  Free  status  if: 

(') 

the condition  In paragraph  A(I) continues  to be  fulfilled. 

( * i ) 

any  animals  introduced  into  the  holding  must  come  from  an  enzootic 

bovine  leucosis  free  holding 

(iii)  all  animals  over  24 months  of  age  continue  to  react  negatively  to a 

test  carried  out  in accordance  with  Chapter  M  at  intervals of  three 

y$ars 

C. The  Leucosis  Free  Status  of  a  holding  shall  be  suspended  if  the  conditions 

detailed  in B  above  have  not  been  complied  with. 

D. The  status  shall  remain  suspended  until  the  following  requirements  are 

complied  with: 

1.  If  a  single  animal  in  an  enzootic  bovine  leucosis  free  holding  has 

reacted  positively  to one of  the  tests  referred  to  in Chapter  II: 

<*6' 

(i) 

the  animal  which  has  reacted  positively,  and,  in  the  case  of  a 

cow,  any  calf  it  may  have  produced,  must  have  left  the  holding 

for 

slaughter 

under 

the 

supervision 

of 

the 

veterinary 

authorities; 

(ii) 

the  remaining  animals  have  reacted  negatively  to  a  serological 

test  carried  out  in  accordance  with  Chapter  II  three  months  at 

least  after  removal  of  the  positive  animal  and  any  possible 

progeny  thereof; 

( M i)  an  epidemiological  enquiry  must  be  conducted  and  the  holdings 

linked  epidemiologically  to  the  infected  holding  must  be 

subjected  to  the measures  laid  down  in  (ii); 

However,  the  competent  authority  may  grant  a  derogation  from  the 

obligation  to  slaughter  the  calf  of  an  infected  cow  where  it  was 

separated  from  its mother  after  calving.  In  this  case,  the  calf  must  be 

made  subject  to  the  requirements  provided  for  in 2  (iii) below. 

2. Where  more  than one  animal  from  an  Enzootic  Bovine  Leucosis  Free  holding 

has  reacted  positively  or  where  infection  has  been  confirmed  in  a 

ho IdIng: 

(i) 

the  animals  which  have  reacted  positively  and  their  calves,  in 

the  case  of  cows,  must  be  removed  for  slaughter  under  the 

supervision  of  the  veterinary  authorities; 

(ii) 

all  animals  aged  over  24  months  must  react  negatively  to  two 

tests  carried  out  in accordance  with  Chapter  II at  an  interval  of 

at  least  4 months  and  less  than  12 months; 

(iii)  all  other  animals  must,  after  identification,  remain  on  the 

holding  until  they  are  aged  over  24 months  and  have  satisfied  the 

tests  referred  to  in  (ii) above. 

(iv) 

an epidemiological  enquiry  must  be  conducted,  and  the  holdings 

linked  epidemiologically  to  the  infected  holding  must  be 

subjected  to  the measures  laid  down  in  (ii) above. 

^ 

However,  the  competent  authority  may  grant  a  derogation  from  the 

obligation  to  slaughter  the  calf  of  an  infected  cow  where  it  was 

separated  from  its mother  after  calving.  In this  case,  the  calf  must  be 

made  subject  to the  requirements  provided  for  in  2(iii). 

3. Where  the  Enzootic  Bovine  Leucosis  Free  status  of  a  holding  has  been 

suspended  for  any other  reason,  al.  animals  in the  holding  aged  over  24 

months  must  give  a  negative  reaction  to  a  serological  test  carried  out 

In accordance with  Chapter  II. 

E.  In accordance  with  the  procedure  in Article  12. the Commission  may  propose 

that  a Member  State or  Region  of  a Member  State may  become  Enzootic  Bovine 

Uucosis  Fre$  if: 

(a)  at  least  99,8  %  of  the  bovine  holdings  are  enzootic  bovine  leucosis 

free holdings within  the meaning  of A above, 

or 

(b)  no  case  of  enzootic  bovine  leucosis  has  been  confirmed  in  the  Member 

State or  region  for  the past  three years and 

in  the  case  of  a  Member  State,  all  animals  aged  over  24  months  in at 

least  10% of  herds,  selected  randomly,  have  been  tested  with  negative 

results  in accordance with Chapter  II  in the previous  24 months or 

in  the  case  of  a  region  or  a  Member  State,  all  animals  aged  over  24 

months  have  undergone  a  test  provided  for  in  Chapter  II  with  negative 

result 

F. A  Member  State  or  a  Region  of  a Member  State  shall  retain  Enzootic  Bovine 

Leucosis  Free status  if: 

(i)  every  year  either  a  random  sample  with  a confidence  rating of  99% has 

established  that  less  than  0,2% of  the  holdings  were  infected  or  not 

less than  20% of  bovine animals over  two years of  age have been  tested 

and  have  reacted  negatively  to  a  test  carried  out  in accordance  with 

Chapter  11. 

or 

(ii) where  no  case  of  enzootic  bovine  leucosis  has  been  recorded  in  the 

Member  State  or  Region  in  a  proportion  of  one  holding  out  of  10  000 

for  at  least  three  years,  a  decision  may  be  taken  in  accordance  with 

Article  12 to cease  routine  serological  testing  provided  that; 

-  all  cattle  slaughtered  within  the  territory  of  that  Member  State  or 

region  are  submitted  to  a  post  mortem  examination  by  an  official 

veterinarian who must  issue notification of  all  tumours with  a view  to 

laboratory  examination,  and. 

-  the  Member  state  shall  report  the occurrence  of  all  cases  of  enzootic 

bovine  leucosis  in  the  area  affected  by  the  decision  to  the 

Commission.  The  Commission  may  according  to  the  procedure  in  Article 

12  propose  that  the  decision  to  cease  routine  serological  testing  be 

suspended  or  revoked  and 

-  any  cattle  which  react  positively  to  an  immune-diffusion  test  are 

slaughtered  and  the  holding  remains  subject  to  restrictions  until  re-

establishment  of  its status pursuant  to Annex  D, Chapter  I, D. 

G.  (i)  The  Enzootic  Bovine  Leucosis  Free  status  of  a  Member  State  or  Region 

of  a Member  state shall  be suspended,  in accordance with  the  procedure 

in Article  12.  if  enzootic  bovine  leucosis  is  detected  and  confirmed 

in more  than  0.2% of  holdings  in the  region or Member  State. 

(i i) The  Enzootic  Bovine  Leucosis  Free  status  may  be  restored,  in 

accordance with  the procedure  in Article  12.  if; 

a.  in  addition  to  the  measures  provided  for  in  paragraphs  D.1  and  D.2 

above,  at  least  20% of  the other  holdings, selected  randomly,  in the 

region or  Member  State  have, within  a  12 month  period, undergone  one 

of  the  tests  referred  to  in Chapter  II. 

b.  the  results  of  this  testing  establish,  with  a  confidence  rating  of 

99%.  that  not  less  than  0.2% of  holdings  are  infected. 

_ -) / -

ANNEX  D 

CHAPTER  II 

TESTS  FOR  ENZOOTIC BOVINE  LEUCOSIS 

Tests  for  enzootic  bovine  leucosis  shall  be  carried  out  by  the  immune-

diffusion  test  under  the  conditions  described  in points  A  and  B  below  or  by 

the  enzyme-linked  immunosorbent  assay  (biisa)  under  the  conditions  described 

in point  C below. The  immune-diffusion  method  may  only  be  used  for  individual 

tests.  If  test  results  are  the  subject  of  a duly-substantiated  challenge, an 

additional  check  shall  be carried out  by means of  the 

immune-diffusion  test. 

A. Agar  gel  immune-diffusion  test  for enzootic bovine  leucosis 

1. The  antigen  to  be  used  in  the  test  must  contain  bovine  leucosis  virus 

glycoproteins. The antigen must  be standardized  against  a standard  serum 

(El  serum)  supplied  by  the  State  Veterinary  Serum  Laboratory, 

Copenhagen. 

2. The  official  institutes  indicated  below  must  be  made  responsible  for 

calibrating  the  standard  working  antigen  of  the  laboratory  against  the 

official  EEC  standard  serum  (El  serum) provided  by  the  State  Veterinary 

Serum  Laboratory,  Copenhagen. 

(a) Germany: 

Bundesforschungsanstalt  fur  Viruskrankheiten  der  Tiere, 

Tub i ngen 

(b) Belgium: 

Institut  national  de  recherches  vétérinaires, Bruxelles 

(c) France: 

Laboratoire  national  de pathologie  bovine, Lyon 

(d) Grand  Duchy  of  Luxembourg:— 

' i/ 

(e)  Italy: 

Istituto ZooprofiI attico Spenmentale,  Perugia 

(f) Netherlands: 

Centraal  Diergeneeskundig  Instituut, Afdeling  Rotterdam 

(g) Denmark: 

Statens Veterinaere Serum  Laboratorium, Copenhagen 

(h)  Ireland: 

Veterinary Research  Laboratory, Abbotstown, Dublin 

(i) United  Kingdom: 

1. Great  Britain: 

The 

Central 

Veterinary 

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, 

(I) Greece: 

Lisboa 

3. The  standard  antigens  used  in  the  laboratory  must  be  submitted  at  least 

once  a  year  to  the  EEC  reference  laboratories  listed  in paragraph  2  above 

for  testing  against  the  official  EEC  standard  serum.  Apart  from  this 

standardization  the antigen  in use can be calibrated  in accordance with B. 

4. The reagents  for  the  test  shall  consist  of: 

(a)  antigen:  the  antigen  must  contain  specific  glycoproteins  of  enzootic 

bovine  leucosis virus which  has been  standardized  against  the official 

EEC  serum; 

(b)  the test  serum; 

(c)  known positive control  serum; 

(d)  Agar  gel, 

0,8% agar, 

8,5%  NaCI, 

0,OS M Tris-buffer  pH 7,2, 

15  ml  of  this  agar  must  be  introduced  into  a  petri  dish  of  85  mm 

diameter,  resulting  in a depth  of  2,6 mm  of  agar. 

5. A  test  pattern  of  seven  moisture-free  wells  be  cut  in  the  agar  to  the 

bottom  of  the  plate;  the  pattern  must  consist  of  one  central  well  and  six 

wells  in a circle  around  it. 

Diameter  of  central  well: 4 mm 

Diameter  of  peripheral  wells: 6 mm 

Distance  between  central  and  peripheral  wells: 3 mm 

6. The  central  well  must  be  filled  with  the  standard  antigen.The  peripheral 

wells  1 and  4  (see diagram  below) are  filled with  the known  positive  serum, 

the wells  2, 3, 5 and  6 with  the  test  sera. The  wells must  be  filled  until 

the meniscus  disappears. 

6 O 

SO 

i 
O 

o 

o 
4 

O 2 

03 

'•»  H-

7. This  results  in  the  following  quantities  being  obtained: 

antigen: 

control  serum: 

test  serum: 

32  fi\ 

73  fi\ 

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 

P OS i 11ve. 

-  r s-

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: 

Petri dish  No  1 
Serum  1:10 

6 < É-

Petri dish  No  2 
Scrum  1:10 

Petri dish  No  3 
Serum  1:5 

Petri dish  No  4 
Serum  1:5 

Add i t i on of ant i gen : 

(i). Petri  dishes  1 and  3 

well  A 

- undiluted  reference antigen, 

well  B 

- 1:2 diluted  reference antigen, 

wells C and  E 

- reference antigen, 

well  D 

« undiluted  antigen  to be tested 

(i i).  Petri  dishes  2 and  4 

well  A 

- undiluted  test  antigen, 

well  B 

«  1:2  diluted  test  antigen, 

well  C 

»  1:4 diluted  test  antigen, 

well  D 

- 1:8 diluted  test  antigen. 

^ 

Additional  Instructions: 

1. The experiment  shall  be carried  out with  two serum  dilutions  (1:5  and 

1:10)  in order' to achieve  optimal  precipitation. 

2.  If the precipitation  diameter  Is too small  with  both  dilutions,  then the 

serum  must  be further  diluted. 

3.  If the precipitation  diameter  in boch  dilutions  is too large  and faint, 

then a  lower  serum  must  be chosen 

4. The final  concentration of the agarose must  be 0,8%;  that of the sera 5% 

and  10% respectively. 

5.  Plot  the measured  diameters  In  the  following  coordinate  system. The 

dilution  of  the  antigen  to be  tested  with  the  same  diameter  as the 

reference  antigen  is the working  dilution. 

Diameter 

13 

12 

11 

-

-

-

10 

-

9  — 

8 

-

7  — 

6 

5 

-

-

4  — 

3 

2 

1 

-

-

-

/ 

' 
1:1 

' 
1:2 

1:3 

I 
1:4 

1:5 

1:6 

1:7 

I 
1:8 

Dilutions  of  anrigens 

">% 

C. Enzvme-linked  immunosorbent  assay  (Elisa)  for  detecting  enzootic  bovine 

ieucosis. 

1. The material  and  reagents  to be used  are as  follows: 

(a) solid  phase microplates,  cuvettes or  any other  solid  phase; 

(b) the  antigen  is  fixed  to  the  solid  phase  with  or  without  the  aid  of 

polyclonal  or  monoclonal  catching  antibodies.  If  antigen  is  coated 

directly  to  the  solid  phase,  all  test  samples  giving  positive 

reactions  have  to be  retested  against  control  antigen  in the case of 

EBL.  The  control  antigen  should  be  identical  to  the  antigen  except 

for  the BLV  antigens.  If catching  antibodies are  coated  to the solid 

phase  the  antibodies  must  not  react  to  antigens  other  than  BLV 

antigens; 

(c) the biological  fluid  to be  tested; 

(d) a corresponding  positive  and  negative  control; 

(e) conjugate; 

(f) a substrate  adapted  to the enzyme  used; 

(g) a stopping  solution,  if necessary; 

(h) solutions  for  the  dilution  of  the  test  samples  for  preparations  of 

the  reagents  and  for  washing; 

(i) a reading  system  appropriate  to the substrate  used. 

S 3-

2.  Standardization  and  sensitivity  of  test: 

The  sensitivity  of  the  Elisa  assay  must  be of  such  a  level  that  E4  serum 

is  scored  positive  when  diluted  10  times  (serum  samples)  or  250  times 

(milk  samples)  more  than  the  dilution  obtained  of  individual  samples 

when  these  are  included  in  pools.  In  assays  where  samples  (serum  and 

milk)  are  tested  individually  E4  serum  diluted  1  to  10  (in  negative 

serum)  or  1  to  250  (in  negative  milk)  must  be  scored  positive  when 

tested  in  the  same  assay  dilution  as  used  for  the  individual  test 

samples.  The  official 

institutes  indicated  in  point  A.2  will  be 

responsible  for  checking  the  quality  of  the  Elisa  method,  and  in 

particular  to  determine,  for  each  production  batch,  the  number  of 

samples  to  be  pooled  on  the  basis  of  the  count  obtained  for  the  E4 

serum. 

The  E4  serum  will  be  supplied  by  the  National  Veterinary  Laboratory, 

Copenhagen. 

3. Conditions  for  use of  the  Elisa  test  for  EBL 

The  Elisa  method  may  be  used  on  a  sample  of  milk  or  whey  taken  from  the 

milk  collected  from  a  farm  with  at  least  30 % of  dairy  cows  in milk. 

If  this  method  is used,  measures  must  be  taken  to  ensure  that  the  samples 

taken  can  be  identified  with  the  animals  from  which  the  milk  or  sera 

examined  were  taken. 

ANNEX  E(l) 

(a) bovine diseases : 

-  Rabies. 

-  Tuberculosis. 

-  Brucellosis, 

-  Contagious Bovine  Pleuropneumonia. 

-  Enzoot i c Bov i ne Leucos i s 

(b) swine  diseases 

-  Rabies. 

-  Bruce 11osis 

ANNEX  EM I) 

Auieszky's  Disease 

Infectious Bovine  Rhinotracheitis 

Brucella  suis  infection 

CERT NO 

SPECIES bovine/swine 

for  slaughter/breeding/production 

MEMBER STATE of origin 

REGION  OF  ORIGIN 

CM-

HOLDING OF ORIGIN 

Name 

ref number 

address 

The holding of origin is; 

The a. imals listed below have been tested  in 

officially  tuberculosis  free 
officially brucellosis  free 
Brucellosis  free 
leucosis free. 

yes/no 
yes/no 
yes/no 
yes/no 

DATE OF DEPARTURE 

Signed 

compliance with Directive 64/432/EEC as 
follows; 

tuberculin test 
yes/not required 
brucellosis SAT  yes/not required 
yes/not required 
leucosis test 

TEST DATE 
.  . 
.  . 
.  . 

Official veterinarian, holding of origin 

ANIMAL  IDENTIFICATION 

total animals 

Breed 

type 

age 

official  identification 

APPROVED MARKET 

LOADING/ASSEMBLY  POINT 

Location 

Address 

Location 

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 
(species/type) destined 

disease for 
/EEC) 

additional guarantees for 
to 

(Com. Decision 

/ 

date health  examination 
expiry date of this cert 

signed 

date 

Name block capitals 

ANNEX  I I 

CORRELATION TABLE 

Updated  Directive 

Directive 64/432/EEC 

Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 

6 1 
e  2.(a) 
e  2.(b) 
e  2.(c) 
e  2.(d) 
e  2.(e) 
e  2.(f) 
e  2.(g) 
e  2.(h) 
e  2.(i) 
e  2.(J) 
e  2.(k) 
e 2.(1) 
e 3.1 
e  3.2.(a) 
e  3.2.(b) 
e  3.2.(c) 
e  3.2.(d) 
e  3.2.(e) 
e 4.1. 
e  4.2 
e 4.3. 
e 5 
e 6.1, first  indent 
e 6.1, second  indent 
e 6.2.(a), 1st  sub 

paragraph 

Article 1 

Article  2.(b) 
Article  2.(c) 
Article  2.(d) 

Article  2.(e) 

Article  2.(f) 
Article  2.(s) 
Article  2.(t) 
Article 2.(1) 
Article  3.1 
Article  3.2.(a) 
Article  3.2.(b) 
Article  3.2.(e) 
Article  3.5 

Article  3.2.(f)(i) 
Article  3.2.(g) 

Article  3.2.(d) 

Article  3.3.(a) 

Art 

e 6.2.(a), 2nd sub 

paragraph 

Art 

e 6.2.(b),  1st sub 

Article  3.3.(b) 

Art 

e 6.2.(b), 2nd sub 

paragraph 

paragraph 

Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 
Art 

6.2.(c) 
6.2.(d) 
6.3. 
7 
8 
9 
10 
11 
12.1. 
12.2. 
12.3. 

Article  3.3.(d) 
Article 3.2.(f)(ii) 

Article 6 

Article 9 
Article  10 

Article 12.1. 
Article 12.2. 
Article 12.3. 

^ ° >-

Updated Direct ive 

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 

Article 12.4., 3rd sub 

paragraph 

Article 13.1. 
Article 13.2. 
Article 13.3. 
Article  13.4., 1st sub 

paragraph 

Article  13.4., 2nd sub 

paragraph 

Article 14 
Article 15 
Annex A 
Annex  B.1.- B.26. 
Annex B.27. 
Annex B.28.- B.31. 
Annex B.32. 
Annex  C.A. 
Annex  C.B. 
Annex C.C. 
Annex  C D. 
Annex C.E. 
Annex  C.F. 
Annex  C.G. 
Annex C.H. 
Annex  D.I. 
Annex D.I I.A.1 - D.I I.A.9 
Annex D.11.A.10 
Annex D.11.B. 
Annex D.11.C. 
Annex  E.I. 
Annex  E.11. 
Annex  F. 

paragraph 

Article 13.1. 
Article 13.2. 
Article 13.3. 
Article 13.4., 1st sub 

paragraph 

Article 14 
Article 16 

Annex B.1.- B.26. 
Annex B.27. 
Annex B.28.- B.31. 
Annex  B.32 
Annex  C.A. 
Annex C.B. 
Annex C.C. 
Annex  C D. 
Annex  C E. 
Annex  C F. 
Annex  C.G. 
Annex G.11.C 
Annex G.I. 
Annex G.11.A.1 - G.I I.A.9 

Annex G.I I.B. 
Annex G. 11 .C 

ISSN 0254-1475 

COM(93) 698 final 

DOCUMENTS 

EN 

10  03 

Catalogue number : CB-CO-93-750-EN-C 

ISBN 92-77-63141-4 

Office for Official Publications of the European Communities 
L-2985 Luxembourg