PROPOSAL FOR A COUNCIL REGULATION ( EEC ) INTRODUCING COMMUNITY MEASURES FOR THE CONTROL OF AVIAN INFLUENZA
Date: 1991-07-30
Institutions: European Commission
Subjects: EU financing, animal disease, health control, poultry farming, veterinary inspection

COMMISSION OF THE EUROPEAN  COMMUNITIES 

COM(91)304  final 

Brussels, 30  July 1991 

Proposal  for a 

COUNCIL REGULATION (EEC) 

introducing Community measures 

for the control of avian  influenza 

(presented by the Commission) 

-  4 ©A4  -

EXPLANATORY  M^MQRANPUM 

Avian  influenza  in  its  highly  pathogenic  form  is  a  contagious  and 

serious  disease  of  poultry.  It  is caused  by  a  virus  which  may  show  a 

great  variation  in pathogenicity  and  in the  clinical  signs  produced  in 

susceptible  birds.  The  disease  has  a  worldwide  distribution  and  the 

virus  can  be  harboured  by  migrating  birds,  in  particular  by  certain 

migratory  waterfowls. 

Article  19 of  Council  Directive 90/539/EEC of  15 October  1990 on  animal 

health  conditions  governing  intra-Community  trade  in, and  imports  from 
third  countries of,  poultry  and  hatching  eggs*1*,  foresee  the  adoption 

of  control  rules  for  combating  avian  influenza before  1 July  1991. 

The  proposed  measures  have  the  aim  of  eradicating  and  preventing  the 

spread  of  avian  influenza  in  the  event  outbreaks  should  occur.  This 

will  be  done  by  "stamping-out"  with  or  without  the  use  of  vaccine  and 

carefully  controlling  the  movement  of  poultry,  poultry  products, 

vehicles  and  any  other  substance  liable  to  transmit  avian  influenza 

virus.  Measures  must  be  introduced  as  soon  as  the  presence  of  avian 

influenza  virus  is suspected  so  that  immediate  and effective  action  can 

be  taken. 

To  ensure  such  actions  the  present  proposal  consists  of  obligations  to 

Member  States, which  include 

to arrange  for  an  investigation  to confirm  or  rule out  the  presence 

of  avian  influenza when poultry  is suspected  of  being  infected; 

to place  holdings  under  surveillance  and  prohibit  movements  to  and 

from  holdings  during  the  surveillance  period  when  avian  influenza 

is suspected; 

to  kill  and  destroy  infected  birds  when  avian  influenza  has  been 

conf i rmed; 

(1) 0J  No L 303, 31.10.1990, p. 6 

-  2 -

to perform  a thorough  epizootiological  inquiry when avian  influenza 

is suspected  and confirmed; 

to establish  protection  zones  (3 km) and surveillance  zones  (10 km) 

around  infected  holdings; 

the  establishment  of 

laboratories  providing 

the  technical 

assistance  necessary  for  correct  implementation  of  disease  control 

measures; 

to  inform  the Commission  about  vaccination  programmes; 

to prepare  a contingency plan. 

-Z  b+o -

PROPOSAL 

for  a 

COUNCIL  REGULATION  (EEC) 

introducing  Community  measures 

for  the  control  of  avian  influenza 

THE  COUNCIL  OF  THE  EUROPEAN  COMMUNITIES, 

Having  regard  to  the  Treaty  establishing  the  European  Economic 

Community,  and  in particular  Article  43  thereof, 

Having  regard  to  the  proposal  from  the  Commission, 

Having  regard  to  the  opinion  of  the  European  Parliament, 

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

Whereas  poultry  is  listed  in  Annex  II  of  the  Treaty;  whereas  the 

marketing  of  poultry  constitutes  an  important  source  of  revenue  for  the 

agricultural  population; 

Whereas,  it  is  necessary  to  establish  at  Community  level  the  control 

measures  to  be  taken  in  the  event  of  outbreak  of  the  highly  pathogenic 

form  of  avian  influenza,  caused  by  an  influenza  virus  with  specific 

characteristics,  and  hereafter  termed  by  avian  influenza  in  order  to 

ensure  national  development  of  the  poultry  sector  and  contribute  to  the 

protection  of  animal  health  in  the  Community; 

Whereas  an  outbreak  of  avian  influenza  can  quickly  take  on  epizootic 

proportions,  causing  mortality  and  disturbances  on  a  scale  liable  to 

reduce  sharply  the  profitability  of  farming  or  poultry  as  a  whole; 

-  2  %UZ 

-

Whereas action must  be  taken  as soon as  the presence  of  the disease  is 

suspected  so  that  immediate  and  effective  control  measures  can  be 

implemented  when  its presence  is confirmed; 

Whereas  it  is necessary  to prevent  any spread of  the disease as soon as 

an  outbreak  occurs,  by  carefully  monitoring  movements  of  animals  and 

the use  of  products  liable  to  be  contaminated,  and  where  appropriate, 

by vaccination; 

Whereas diagnosis of  the disease must  be carried out under  the auspices 

of  a responsible  laboratory,  the  coordination  of  which  must  be ensured 

by a Community  reference  laboratory; 

Whereas common measures  for  the control  of avian  influenza  form a basis 

for maintaining  a uniform  standard of animal  health; 

Whereas  it  is  appropriate  to  confer  upon  the  Commission  the  task  of 

taking  the necessary  applicatory measures; 

HAS ADOPTED THIS REGULATION: 

A r t i c le 1 

1.  This  Regulation  defines  the  Community  control  measures  to  be 

applied  in  the  event  of  an outbreak  of  avian  influenza  in poultry 

without  prejudice  to  the  Community  provisions  governing  intra-

Community  trade. 

2.  This regulation shall  not  apply,  in the event: of detection of avian 

influenza  in  other  birds,  however,  in  this  case,  Member  States 

shall  inform  the Commission  of any measure  they take. 

-  3 -

Article  2 

1.  For  the  purpose  of  this  Regulation,  the  definitions  given  in 

Article  2  of  Council  Directive  90/539/EEC  on  animal  health 

conditions  governing  intra-Community  trade  in,  and  imports  from 

third  countries  of  poultry  and  hatching  eggs( 1)  shall  apply  as 

appropriate. 

2.  The following definitions shall  also apply: 

(a) "infected  poultry" means any poultry: 

in which  the presence of avian  influenza,  in the meaning of 

Annex  I,  has  been  officially  confirmed  following  an 

examination  by an approved  laboratory, or 

in  the  case  of  second  and  subsequent  outbreaks  in  which 

clinical  signs or post-mortem  lesions consistent  with  avian 

influenza are present; 

(b) "poultry  suspected of being  infected" means any poultry  showing 

clinical  signs  or  post-mortem  lesions  which  are  such  that  the 

presence of avian  influenza may  reasonably  be suspected; or  the 

presence  of  influenza  A  virus  of  subtype  H5  or  H7  has  been 

demonstrated. 

(c) "poultry  suspected  of  being  contaminated"  means  any  poultry 

which may  have been directly or  indirectly exposed  to the avian 

influenza  virus,  or  influenza  A  virus  of  H5  subtype  or  H7 

subtype. 

(d) "competent  authority":  veterinary  authority  appointed  for  the 

purpose  by  the  national  administration  of  the  country 

concerned,  being  directly  responsible  to  the  national 

administration  within  the  scope  of  this  Regulation,  and 

reporting  through  the national  administration. 

(e) "official  veterinarian":  the  veterinarian  designated  by  the 

competent  authority of  the Member  State. 

(1) OJ No L 303, 31.10.1990, p. 6 

_ 4 -

Article 3 

Any  suspected  case of  avian  influenza  shall  be  immediately  notified  to 

the competent  authority. 

Article 4 

1.  When  poultry  are  suspected  of  being  infected  or  contaminated  with 

avian  influenza,  the  official  veterinarian  shall  immediately 

arrange an  investigation to confirm or  rule out  the presence of the 

disease  and,  in  particular,  must  take  or  have  taken  the  samples 

necessary  for  laboratory  examination. 

2.  As  soon  as  the  suspected  infection  is  notified,  the  competent 

authority  shall  have  the holding  placed under official  surveillance 

and shall  in particular  require  that: 

(a) a  record  be  made  of  all  categories  of  poultry  on  the  holding 

showing  in  respect  of  each  of  the  categories  the  numbers  of 

poultry  which  have  died,  which  show  clinical  signs,  and  which 

show  no  signs.  The  record  shall  be  kept  up-to-date  and  be 

produced on  request  and may be checked  at each  visit; 

(b) all  poultry  on  the  holding  must  be  kept  in  their  living 

quarters  or  some  other  place  where  they  can  be  isolated  and 

without  contact  with other birds; 

(c) no poultry enter or  leave the holding; 

(d) all  movement 

of  persons,  other  animals  and  vehicles  to  or  from  the 

ho Id i ng, 

of  poultry  meat  or  carcasses,  or  of  animal  feed, 

implements,  waste  and  litter  or  any  other  thing  liable  to 

transmit  avian  influenza  virus  from  the holding 

be prohibited unless authorized  by the official  veterinarian; 

- 5 -

(e) no  eggs  shall  leave  the  holding  with  the  exception  of  table 

eggs  which  have  been  disinfected  to  the  satisfaction  of  the 

official  veterinarian; 

(f) appropriate means of  disinfection  be used  at  the entrances  and 

exits of buildings housing poultry  and of  the holding  itself; 

(g) an  epizootiological  inquiry  be  carried  out  in  accordance  with 

Article 7. 

3.  Until  such  time  as  the official  measures  laid  down  in paragraph  2 

are  enforced,  the owner  or  keeper  of  any  poultry  in which  disease 

is suspected  shall  take all  reasonable  action  to ensure  compliance 

with paragraph  2, particularly  indents  (b), (c), (d) and (e). 

4.  The  competent  authority  may  apply  any of  the measures  provided  for 

in  paragraph  2  to  other  holdings  should  their  location,  their 

configuration,  or  contacts  with  the  holding  where  the  disease  is 

suspected  give reason  to suspect  possible contamination. 

5.  The  measures  referred  to  in  paragraphs  2,  3  and  4  shall  not  be 

withdrawn until  the suspicion of avian  influenza  has been  ruled out 

by the official  veterinarian. 

Article 5 

1.  As  soon  as  the  presence  of  avian  influenza  has  been  officially 

confirmed on a holding,  the competent  authority,  in addition  to the 

measures  listed  in  Article  4(2)  shall  require  the  following 

measures to be undertaken: 

(a) all  poultry on the holding  shall  without  delay  be killed on the 

spot.  The  poultry  which  have  died  or  have  been  killed  and all 

eggs  shall  be destroyed.  These operations  shall  be  carried  out 

in a way which minimizes  the risk of spreading  disease; 

- 6 -

(b) any  substance or  waste, such  as animal  feed,  litter  or  manures 

liable  to be contaminated,  shall  be destroyed  or  treated;  this 

treatment,  carried  out  in  accordance  with  the  instructions  of 

the official  veterinarian, shall  ensure  the destruction, of any 

avian  influenza  virus present; 

(c) where  poultry  have  been  slaughtered  during  the  presumed 

incubation  period  of  disease  the meat  from  those  poultry  shall 

wherever  possible be traced and  destroyed; 

(d) hatching  eggs  laid  during  the presumed  incubation  period  which 

have been moved  from the holding shall  be traced  and  destroyed; 

but  poultry  which  have  already  hatched  from  the  eggs  shall  be 

placed  under  official  surveillance;  table  eggs  laid  during  the 

presumed  incubation  period  which  have  been  moved  from  the 

holding  shall  wherever  possible be traced  and  destroyed; 

(e) after  carrying  out  operations  listed  in  sub-paragraph  (a) the 

buildings  used  for  housing  poultry,  their  surroundings,  the 

vehicles  used  for  transport  and  all  equipment  likely  to  be 

contaminated  shall  be  cleaned  and  disinfected  in  accordance 

with  the provisions of Article 11; 

(f) no  poultry  shall  be  reintroduced  to the  holding  until  at  least 

21  days  after  completion  of  operations  provided  for  in  sub 

paragraph (e); 

(g) an epizootiological  inquiry  shall  be carried  out  in accordance 

with Article 7. 

2.  The  competent  authority  may  extend  the  measures  provided  for  in 

paragraph  1  to  other  holdings  should  their  location,  their 

configuration,  or  contact  with  the  holding  where  the  disease  has 

been confirmed given  reason  to suspect  possible contamination. 

- 7 -

Article  6 

1.  In  the  case  of  holdings  which  consist  of  two  or  more  separate 

flocks  the  competent  authority  may  grant  a  derogation  from  the 

requirements  of  Article  5(1)(a),  for  healthy  flocks  of  a  holding 

which  is  infected,  provided  that  the  official  veterinarian  has 

confirmed  that  the  operations  carried  out  there  are  such  that  the 

flocks  are  completely  separate  as  regards  housing,  keeping  and 

feeding,  so  that  the  virus  cannot  spread  from  one  flock  to  another. 

2.  The  Commission  shall  in accordance  with  the  procedure  in Article  21 

lay  down  the  criteria  to  be  applied  for  granting  a  derogation  as 

referred  to  in paragraph  1. 

Article  7 

1.  The  epizootiological  inquiry  shall  deal  with: 

the  length  of  time  during  which  avian  influenza  may  have 

existed  on  the  holding; 

the  possible  origin  of  the  avian  influenza  on  the  holding  and 

the  identification  of  other  holdings  on  which  there  are  poultry 

which  may  have  become  infected  or  contaminated  from  the  same 

source; 

the  movement  of  persons,  poultry  or  other  animals,  vehicles, 

eggs,  meat  and  carcasses  and  any  implement  or  substance  likely 

to  have  carried  avian  influenza  virus  to or  from  the  holding  in 

question. 

2.  In order  to  provide  full  coordination  of  all  measures  necessary  to 

ensure  eradication  of  avian  influenza  as  quickly  as  possible  and 

for  the  purpose  of  carrying  out  the  epidemiological  inquiry,  a 

crisis  unit  shall  be  be  established. 

(3) 

- 8 -

The  general  rules  concerning  National  Crisis  units  and  Community 

crissis units are as  laid down  in Council  Regulation  ../.../EEC. 

Article 8 

1.  Where  the official  veterinarian  has  reason  to suspect  that  poultry 

on  any  holding  may  have  been  contaminated  as  a  result  of  the 

movement  of  persons, animals or  vehicles or  in any other  way,  that 

holding  shall  be  placed  under  official  control  in  accordance  with 

paragraph 2. 

2.  The purpose of  the official  control  shall  be to detect  immediately 

any  suspicion  of  avian  influenza,  count  the  poultry  and  monitor 

their movements and, where appropriate,  to take the action  provided 

for  in paragraph 3. 

3.  When  a  holding  is  subject  to  the  official  control  under  the 

provisions  of  paragraph  2,  the  competent  authority  shall  prohibit 

removal  of  poultry  from  the  holding  other  than  for  transport 

directly  to  a  slaughterhouse  under  official  supervision  for  the 

purpose  of 

immediate 

slaughter.  Prior 

to  granting 

such 

authorization,  the  official  veterinarian  must  have  carried  out  a 

clinical  examination  of  all  the  poultry  to  exclude  presence  of 

avian  influenza  on  the  holding.  The  above  movement  restrictions 

shall  be  imposed  for  a  period  of  21  days  from  the  latest  date  of 

potential  contamination.  However,  such  restrictions  must  apply  for 

a period of at  least 7 days. 

4.  Where  it considers  that  conditions permit,  the  competent  authority 

may  limit  the measures  provided  for  in  this  article  to  a  part  of 

the holding and  to the poultry  contained  therein, provided  that  the 

poultry  there  have  been  housed,  kept  and  fed  completely  separately 

by separate staff. 

- 9 

Article  9 

1.  Once  the  diagnosis  of  avian  influenza  has  been  officially 

confirmed,  the  competent  authority  shall  establish  around  the 

infected  holding an  infected area  involving a protection zone based 

on  a  minimum  radius  of  3  km  and  a  surveillance  zone  based  on  a 

minimum  radius of  10 km. The establishment of  these zones must  take 

account  of natural  boundaries and  the epidemiology  of  the outbreak. 

2.  The measures applied  in the protection zone shall  include: 

(a) the  identification  of  all  holdings  having  poultry  within  the 

zone ; 

(b) periodic  visits  to all  the  holdings  having  poultry,  a clinical 

examination  of  those  poultry  including,  if  necessary,  the 

collection  of  samples  for  laboratory  examination;  a  record  of 

visits and  findings must  be kept; 

(c) the  keeping  of  all  poultry  in  their  living  quarters  or  some 

other  place where  they can be  isolated; 

(d) the  use  of  appropriate  means  of  disinfection  at  the  entrances 

and exits of  the holding; 

(e) the  control  of  movements  of  persons  handling  poultry,  poultry 

carcasses and eggs and vehicles carrying  poultry, carcasses and 

eggs  within  the  zone;  in  general  transport  of  poultry  is 

prohibited  except  for  transit  by major  highways or  railways; 

(f) a  prohibition  on  removing  poultry  and  hatching  eggs  from  the 

holding  on  which  they  are  kept  unless  the  competent  authority 

has authorized  the  transport: 

-  10 -

(I) 

of  poultry  for  immediate  slaughter  to a slaughterhouse 

preferably  located  in the  infected  area or,  if  that  is 

not  possible,  to  a  slaughterhouse  designated  by  the 

competent  authority  outside  the  infected  area.  The 

special  health  mark  provided  for  in  Article  6(1)  of 

Regulation  ../ 

/EEC  on  animal  health  conditions 

governing  intra-Community  trade  in  and  imports  from 

third  country  of  fresh  poultry  meat  and  fresh  meat  of 

reared  game  birdsd)  must  be  applied  to  this  poultry 

meat; 

(ii) 

of  day-old  chicks or  ready  to  lay pullets  to a holding 

within  the  infected  area  at  which  there  are  no  other 

poultry. This holding must  be placed under  the official 

control  provided  for  in Article 8, paragraph (2); 

(iii)  of  hatching  eggs  to  a  hatchery  situated  inside  the 

infected area or one outside  the area designated  by the 

competent  authority;  before  dispatch  eggs  and  their 

packing must  be disinfected; 

Movements  allowed  in  (i),  (ii)  and  (iii)  shall  be  directly 

executed, under official  control. They shall  be authorized  only 

after  the  official  veterinarian  has  carried  out  a  health 

inspection  of  the  holding. The means of  transport  used  must  be 

cleaned  and disinfected  before and after use. 

(g) a prohibition on removing or spreading  poultry manure or  litter 

without  authorization; 

(h) the prohibition of  fairs, markets, shows or other  gatherings of 

poultry or other birds. 

3.  The measures applied  in the protection  zone shall  be maintained  for 

at  least  21  days  after  the  carrying  out,  of  preliminary  cleaning 

and  disinfection  operations  on  the  infected  holding  in  accordance 

with  Article  11. The  protection  zone  shall  thereafter  be  part  of 

the surveillance zone. 

(1) COM  (89) 507 final 

- 11 -

4.  The measures applied  in the surveillance zone shall  include: 

(a) the  identification  of  all  holdings  having  poultry  within  the 

zone; 

(b) the  control  of  poultry  and  hatching  egg  movement  within  the 

zone ; 

(c) a prohibition on the movement of poultry out of  the zone during 

the  first  15  days,  except  for  movement  directly  to  a 

slaughterhouse outside  the surveillance  zone  designated  by  the 

competent  authority.  The  special  health  mark  provided  for  in 

Article 6 of Regulation  ../..../EEC on animal  health conditions 

governing  intra-Community  trade  and  imports  from  third 

countries  of  fresh  poultry  meat  and  fresh  meat  of  reared  game 

birds must be applied  to this poultry meat; 

(d) a  prohibition  on  the  movement  of  hatching  eggs  out  of  the 

surveillance  zone  unless  to  a  premises  designated  by  the 

competent  authority. Before dispatch  the eggs and  their packing 

must be disinfected; 

(e) a prohibition on  the movement  of  poultry  manure and  litter out 

of  the zone; 

(f) a  prohibition  of  fairs, markets, shows or  other  gatherings  of 

poultry  and other birds; 

(g) without  prejudice  to provisions of  (b) and  (c) the  prohibition 

of  transport  of  poultry, except  for  transit  by  major  highways 

or  raiI ways. 

5.  The  measures  applied  in the  surveillance  zone  shall  be  maintained 

for at  least 30 days after  the carrying out of preliminary cleaning 

and  disinfection  operations  on  the  infected  holding  in  accordance 

with Article 11. 

-  12  -

Article  1Q 

1.  The  competent  authority  shall  determine  the  arrangements  allowing 

them  to  trace  the movement  of  eggs  and  poultry. 

2.  The  owner  or  keeper  of  poultry  shall  be  required  to  supply  the 

competent  authority,  on  request  by  that  authority,  with  information 

concerning  poultry  and  eggs entering or  leaving  his  holding. 

3.  All  persons  engaged  in  the  transport  or  marketing  of  poultry  and 

eggs  shall  be  able  to  supply  the  competent  authority  with 

information  concerning  the movements  of  poultry  and  eggs  which  they 

have  transported  or  marketed  and  to  furnish  all  the  details 

concerning  such  information. 

A r t i c le  11 

1.  The  disinfectants  to  be  used  and  their  concentrations  shall  be 

approved  by  the  competent  authority. 

2.  The  cleaning  and  disinfection  operations  shall  be  carried  out  under 

official  supervision,  in accordance  with  instructions  given  by  the 

official  veterinarian. 

Article  12 

Collection  of  samples  and  laboratory  testing  to  detect  the  presence  of 

avian  influenza  virus  shall  be  carried  out  in accordance  with  Annex  I. 

Art'Cle  13 

1.  Each Member  State  shall  designate: 

(a) one  or  more  national  laboratories  at  which  facilities  and 

expert  personnel  shall  be  maintained  to  permit  assessment  of 

the  pathogenicity  of  influenza  virus  isolates  (Annex  I Chapter 

7)  and  identification  of  influenza  A  viruses  of  H5  or  H7 

subtypes;. 

-  13 

(b) one or more  national  laboratories  at which  reagents  for  use  in 

regional  laboratories are  tested; 

(c) one  or  more  national  institutes  or  laboratories  at  which 

authorized  vaccines  may  be  tested  in  order  to  verify  their 

conformity  with  the specifications  laid down  in the  marketing 

authorization. 

2.  The  national  laboratories  listed  in  Annex  II  are  responsible  for 

coordinating  standards  and  methods  of  diagnosis,  use  of  reagents 

and  testing of vaccines. 

3.  The  national  avian  influenza  laboratories  referred  to  in paragraph 

2  shall  be  responsible  for  coordinating  the  standards  and 

diagnostic  methods  laid  down  in  each  avian  influenza  diagnostic 

laboratory within  the Member  State. To this end: 

(a) they may provide diagnostic reagents  to regional  laboratories; 

(b) they  shall  control  the quality of  all  diagnostic  reagents used 

in that Member  State; 

(c) they shall  arrange comparative  tests periodically; 

(d) they  shall  hold  isolates  of  avian  influenza  virus  from  cases 

confirmed  in that Member  State; 

(e) they shall  ensure  the confirmation of positive  results obtained 

in regional  diagnostic  laboratories. 

4.  The  national  laboratories  listed  in Annex  II shall  liaise with  the 

Community  reference  laboratory  referred  to  in Article 14. 

Article 14 

The  Community  reference  laboratory  of  avian  influenza  is mentioned  in 

Annex  III. The powers and  duties of  the  laboratory  shall  be  laid down, 

in  so  far  they  are  not  already  covered  by  Article  28  of  Council 

Decision  90/424/EEC(1^  on  expenditure  in  the  veterinary  field,  in 

accordance with  the procedure  laid down  in Article 21. 

(1) OJ No L 224, 18.08.1990, p. 19 

-  14 -

Article 15 

1.  Vaccination  against  avian  influenza  with  vaccines  authorized  by  the 

competent  authority  may  be  used  only  to  supplement  the  control 

measures  carried  out  when  the disease  appears. 

2.  The  decision  to  introduce  vaccination  to  supplement  control 

measures  shall  be  taken  by  the Commission  in collaboration  with  the 

Member  State  concerned,  acting  in  accordance  with  the  procedure 

laid  down  in Article  21. This  decision  shall  have  particular  regard 

to: 

the  concentration of  poultry  in the  affected  area; 

the  characteristics  and  composition  of  the  vaccine  to be  used: 

the procedures  for  supervision  of  the distribution,  storage  and 

use of  vaccines; 

the  species  and  categories  of  poultry  which  shall  be  subject  to 

vaccination; 

the  areas  in which  vaccination  shall  be  carried  out. 

3.  Where  a  Member  State  is  authorized,  in  accordance  with  the 

provisions  of  paragraph  2,  to  have  recourse  to  emergency 

vaccination  on  a  limited  part  of  its  territory  the  status  of  the 

remainder  of  the  territory  shall  not  be  affected,  provided  that  the 

immobilization  measures  for  the  vaccinated  animals  are  effective 

during  a  period  of  3  months  following  the  end  of  the  vaccination 

operations. 

Article  16 

1.  When,  if  a  given  region,  an  epizootic  of  avian  influenza  is 

exceptionally  serious  and  is  tending  to  spread,  the  Member  States 

concerned: 

shall  declare  a  demarcated  territorial  area  including  at  least 

all  the  protection  and  surveillance  zones  in  that  area  to  be  a 

"high  health  risk  area"; 

-  15  -

shall  apply  the  measures  provided  for  In  Article  9(3)  in  the 

"high  health  risk  area"; 

shall  prohibit  all  live  poultry  and  hatching  eggs  from  leaving 

the  "high  health  risk  area"; 

shall  inform  the  Commission  and  other  Member  states  within  the 

framework  of  the  Standing  Veterinary  Committee  about  disease 

situation  and  appI led  control  measures. 

2.  The  boundaries of  the  "high  health  risk  area" may  be  revised  with  a 

progressive  elimination  of  surveillance  zones.  The  measures  laid 

down  in  the  preceding  paragraph  shall  be  discontinued  after  the 

elimination  of  the  last  surveillance  zone. 

3.  If an  exceptionally  serious  situation  continues,  the measures  to  be 

taken  by  the  Member  States  concerned,  in  particular  determination 

of  the  "high  health  risk  area"  and  recourse  to  the  provisions  of 

Article  15,  must  be  decided  in  accordance  with  the  procedure  laid 

down  in Article  21. 

Article 17 

1.  Each  Member  State  shall  draw  up  a  contingency  plan,  specifying  the 

national  measures  to  be  implemented  in  the  event  of  an  outbreak  of 

avian  influenza. 

This  plan  should  allow  access  to  facilities,  equipment,  personnel 

and  all  other  appropriate  materials  necessary  for  the  rapid  and 

efficient  eradication  of  the  outbreak.  It  must  give  a  precise 

indication  of  the  vaccine  requirements  which  each  Member  State 

concerned  considers  it needs  in the event  of  emergency  vaccination. 

- 16 -

2.  The  criteria  to  be  applied  for  drawing  up  the  contingency  plan 

shall  be  those  laid  down  in Commission  Decision  91/42/EEC(1) of  8 

January  1991  laying down  the criteria  to be applied when drawing up 

contingency  plans  for  the  control  of  foot  and  mouth  disease,  in 

application  of  Article  5  of  Council  Directive  90/423/EEC,  which 

shall  apply mutatis mutandis. 

The  Commission  may,  in  accordance  with  Article  21,  amend  or 

supplement  those  criteria  taking  into  account  the  specific  nature 

of avian  influenza. 

3.  Plans  drawn  up  in  accordance  with  the  criteria  provided  for  in 

paragraph  2 shall  be submitted  to the Commission  not  later  than  12 

months after  this regulation enters  into force. 

4.  The  Commission  shall  examine  the  plans  in  order  to  determine 

whether  they  permit  the desired  objective  to be attained  and  shall 

suggest  to  the  Member  State  concerned  any  amendments  required  in 

particular  to  ensure  that  they  are  compatible  with  those  of  the 

other Member  States. 

The  Commission  shall  approve  the  plans,  if  necessary  amended,  in 

accordance with  the procedure  laid down  in Article 21. 

The  plans  may  subsequently  be  amended  or  supplemented,  in 

accordance  with  the  same  procedure,  to  take  into  account 

developments  in the situation. 

ArtiCie  18 

Veterinary  experts  from  the  Commission  may,  in collaboration  with  the 

authorities  of  the  concerned  Member  State,  insofar  as  is necessary  to 

ensure uniform  application of  this Regulation, make on-the-spot checks-, 

the  Commission  shall  inform  the  Member  States  of  the  results  of  the 

investigation. 

(1) OJ No L  23, 29.01.1991, p. 29 

-  17 -

A  Member  State  in whose  territory  a  check  is being  carried  out  shall 

give  all  the necessary  assistance  to the experts  in carrying  out  their 

duties. 

The  general  provisions  for  implementing  this  Article  shall  be 

determined  in accordance with  the procedure  laid down  in Article 21. 

Article  19 

Modalities  for  financial  participation of  the Community  to  the  actions 

arising  from  this  Regulation  are  fixed  in Council  Decision  90/424/EEC 

on expenditure  in the veterinary  field. 

Article 2Q 

The  Annexes  to  this  Regulation  may  be  amended  by  the  Commission,  in 

accordance with  the procedure  laid down  in Article 21,  in particular  in 

order  to take  into account  the development  in diagnostic procedures. 

Article 21 

1.  The  Commission  shall  be  assisted  by  the  Standing  Veterinary 

Committee,  hereinafter  referred  to  as  "the  Committee";  set  up  by 

Decision  68/361/EEC<1). 

2.  Where  the  procedure  laid  down  in  this  Article  is  to  be  followed, 

the following  provisions shall  apply. 

The  representative of  the Commission  shall  submit  to  the  Committee 

a  draft  of  the  measures  to  be  taken.  The  Committee  shall  deliver 

its opinion on the draft  within a time  limit which  the Chairman may 

lay  down  according  to  the urgency  of  the matter,  if  necessary  by 

taking  a vote. 

The  opinion  shall  be  recorded  in  the  minutes;  in  addition,  each 

Member  State  shall  have  the  right  to  ask  to  have  its  position 

recorded  in the minutes. 

(1) OJ No L 255,  18.10.1968, p. 23 

-  18 -

The  Commission  shall  take  the  utmost  account  of  the  opinion 

delivered  by  the  Committee.  It  shall  inform  the  Committee  of  the 

manner  in which  its opinion  has  been  taken  into  account. 

Article 22 

This  Regulation  shall  enter  into  force  on  1 July  1991. 

This  Regulation  shall  be  binding  in  its  entirety  and  directly 

applicable  in all  Member  States. 

Done  at  Brussels 

For  the  Council 

ANNEX I 

DIAGNOSTIC PROCEDURES  FOR THE  CONFIRMATION 

AND DIFFERENTIAL DIAGNOSIS OF AVIAN  INFLUENZA  (AI) 

The  following  procedures  for  the  isolation  and  characterization  of 

avian  influenza viruses should be regarded  as guidelines and  the minima 

to be applied  In the diagnosis of  the disease. 

For  the  purpose  of  the  diagnostic  procedures  for  the  confirmation  and 

differential  diagnosis  of  avian  influenza  the  following  definition 

shall  apply. 

"Avian  influenza" means an  infection of poultry  caused by any  influenza 

A  virus  which  has  an  intravenous  pathogenicity  index  in  six-week-old 

chickens greater  than  1.2 or  any  infection with  influenza  A  viruses of 

H5 or  H7  subtype  for  which  nucleotide  sequencing  has  demonstrated  the 

presence  of  multiple  basic  amino  acids  at  the  cleavage  site  of  the 

haemagglutinin. 

CHAPTER  1: Sampling  and  treatment  of  samples 

1.  Samples 

Cioacal  swabs  (or  faeces)  and  tracheal  swabs  from  sick  birds; 

faeces or  intestinal  contents, brain  tissue, trachea,  lungs,  liver, 

spleen  and  other  obviously  affected  organs  from  recently  dead 

birds. 

2.  Treatment of samples 

The  organs  and  tissues  listed  above  may  be  pooled,  but  separate 

treatment  of  faecal  material  is essential.  Swabs  should  be  placed 

in  sufficient  antibiotic  medium  to  ensure  full  immersion.  Faeces 

samples and organs should  be homogenised  (in an enclosed  blender or 

using  a  pestle  and  mortar  and  sterile  sand)  in  antibiotic  medium 

and  made  to  10-20% w/v  suspensions  in  the medium.  The  suspensions 

should  be  left  for  about  two  hours  at  ambient  temperature  (or 

longer  periods "at  4*C) and  then  clarified  by  centrifugation  (eg. 

800 to 1000 x g for  10 minutes). 

-  20-

3.  Antibiotic  medium 

Different  laboratories  have  used  various  formulations  of  antibiotic 

medium  with  success  and  National  Laboratories  will  be  able  to offer 

advice  for  a particular  country.  High  concentrations of  antibiotics 

are  required  for  faeces  amples  and  a typical  mixture  is: 

10,000  units/ml  penicillin,  10  mg/ml  streptomycin,  0.25  mg/ml 

gentamycin  and  5,000  units/ml  mycostatin  in  phosphate  buffered 

saline.  These  levels  can  be  reduced  up  to  five-fold  for  tissues  and 

tracheal  swabs.  For  control  of  Chlamydia  organisms  50  mg/ml 

oxytetracycline  may  be  added.  It  is  imperative  when  making  the 

medium  that  the  pH  is checked  after  the  addition  of  the  antibiotics 

and  readjusted  to pH  7.0-7.4. 

CHAPTER  2:  Virus  Isolation 

Virus  isolation  in embryonated  fowls'  eggs 

The  clarified  supernatant  fluid  should  be  inoculated  in  0.1-0.2  ml 

amounts  into  the  allantoic  cavity  of  each  of  a  minimum  of  four 

embryonated,  fowls'  eggs  which  have  been  incubated  for  8-10  days. 

Ideally,  these  eggs  should  be  obtained  from  a  specific  pathogen  free 

flock,  but  when  this  is  impracticable  it  is  acceptable  to  use  eggs 

obtained  from  a  flock  shown  to  be  free  of  antibodies  to  Avian 

Influenza.  The  inoculated  eggs  are  held  at  37*C  and  candled  daily.  Eggs 

with  dead  or  dying  embryos  as  they  arise,  and  all  remaining  eggs  six 

days  after  inoculation  should  be  chilled  to  4*C  and  the  allantoic-

amniotic 

fluids  tested 

for  haemagglunination  activity. 

If  no 

haemagglutination  is  detected  the  above  procedure  is  repeated  using 

undiluted  allantoic/amniotic  fluid  as  inoculum. 

When  haemagglutination  is  detected  the  presence  of  bacteria  should  be 

excluded  by  culture.  If  bacteria  are  present  the  fluids  may  be  passed 

through  a  450nm  membrane  filter,  further  antibiotics  added  and 

inoculated 

into 

embryonated 

eggs 

as 

above. 

-Ii-

CHAPTER  3;  Differential  diagnosis 

1.  Preliminary  differentiation 

Because  it  Is  Important  that  control  measures  aimed  at  limiting  the 

spread  of  virus  should  be  implemented  as  soon  as  possible,  each 

regional  laboratory  should  be  in  a  position  to  identify  any 

isolated  haemagglutinating  virus  as  influenza  viruses  of  H5  or  H7 

subtype 

in 

addition 

to 

Newcastle 

disease 

virus. 

The 

haemagglutinating  fluids  should  be  used  in  a  haemagglutination 

inhibition  test  as  described  in  Chapters  5  and  6.  Positive 
inhibition  ie.  24,  or  more,  with  polyclonal  antisera  specific  for 
H5  or  H7  subtypes  of  influenza  A  and  of  a  titre  of  at  least  29 

would  serve  as  preliminary  identification  enabling  the  imposition 

of  interim  control  measures. 

2.  confirmatory  identification 

Since  there  are  13  haemagglutinin  subtypes  and  9  neuraminidase 

subtypes  of  influenza  viruses  and  variations  occur  within  each  of 

these  it  is  not  practicable  nor  cost  effective  for  each  national 

laboratory  to  hold  antisera  which  will  allow  full  antigenic 

characterization  of  influenza  isolates.  However,  each  national 

laboratory  should: 

(i) 

confirm  that  the  isolate  is  an  influenza  A  virus  using  an 

immunodoublediffusion  test  to  detect  group  antigen  as 

described  in Chapter  9 of  this Annex  (immunofluorescence  or 

EL I SA  techniques  to  detect  group  antigens  may  be  used  if 

preferred  by  the national  laboratory); 

(Ii) 

determine  whether  or  not  the  isolate  is  of  H5  or  H7 

subtype; 

-Il-

(iii)  carry  out  an  intravenous  pathogenicity  index  test  in six-

week-old  chickens  as described  in Chapter  7 of  this Annex. 

Intravenous  pathogenicity  indices  of  greater  than  1.2 

indicate  the  presence  of  virus  requiring  the  full 

implementation  of  control  measures  (It  would  be  a  useful 

exercise  if national  laboratories also carried out  tests to 

determine  the capacity  of  an  isolate  to produce  plaques  in 

cell  cultures as specified  In Chapter 8 ). 

National  laboratories should  immediately  submit all  avian  influenza 

and  all  H5  and  H7  isolates  to  the  Community  Reference  Laboratory 

for  full  characterization. 

3.  Further  typing and characterization of  isolates 

The 

Community 

Reference 

Laboratory 

should 

receive 

all 

haemagglutinating  viruses  from  the  national  laboratories  for 

further  antigenic  and  genetic  studies  to  enable  a  greater 

understanding  of  the  epizootioiogy  of  the  disease(s)  within  the 

European  Community  in keeping  with  the  functions and  duties of  the 

reference  laboratory. 

In  addition  to  these  duties  the  Community  Reference  Laboratory 

shall  carry  out  full  antigenic  typing  for  all  influenza  viruses 

received.  For  H5  and  H7  viruses  which  do  not  have  intravenous 

pathogenicity  indices  greater  than  1.2,  nucleotide  sequencing  of 

the  haemagglutinin  gene  to  determine  whether  or  not  there  are 

multiple  basic  animo  acids  at  the  cleavage  site  of  the 

haemagglutinin protein should also be carried out. 

-11-

CHAPTER  4;  Serological  tests  for  avian  jnfluenza 

virus  antibodies 

During  eradication  programmes  where  the  H  subtype  of  the  virus 

responsible  is  already  known,  or  by  using  the  homologous  virus  as 

antigen,  serological  monitoring  for  evidence  of  infection  may  be 

done  using  haemaggluniation  inhibition  tests  as  described  in 

Chapters  5  and  6. 

If  the  haemagglutinin  subtype  is  not  know,  evidence  for  infection 

with  influenza  A  viruses  may  be  obtained  by  detecting  antibodies 

directed  to  the  group  specific  antigens.  For  this  purpose  either  an 

immunodoublediffusion  test  (as  described  in  Chapter  9  of  this 

Annex)  or  an  ELISA  test  may  be  used  (a  problem  with  ELISA  is  the 

host  specificity  of  the  test  since  it  is dependent  on  the  detection 

of  host  immunoglobulins).  Waterfowl  rarely  give  positive  results  in 

immunodoublediffusion  tests  and,  unless  the  subtype  is known,  it  is 

probably  only  practicable  to examine  such  birds  for  the  presence  of 

antibodies  to  H5  and  H7  subtypes. 

(a) samples 

Blood  samples  should  be  taken  from  all  birds  if  the  flock  size 

is  less  than  20  and  from  20  birds  from  larger  flocks  (This  will 

give  a  >99%  probability  of  detecting  at  least  one  positive 

serum  if  25%  or  more  of  the  flock  is  positive,  regardless  of 

flock  size).  The  blood  should  be  allowed  to  clot  and  serum 

removed  for  testing. 

(b) Examination for antibodies 

Individual  serum  samples  should  be  tested  for  their  ability  to 

inhibit  influenza  virus  haemagglutinating  antigen  in  standard 

haemagglut(nation  inhibition  tests  as  defined  in Chapter  6. 

-Ik-

There  is  some  debate  as  to  whether  4  or  8  haemagglut inin  units 

should  be  used  for  the  HI  tests.  It  would  appear  that  either  is 

valid  and  the  choice  should  be  left  to  the  discretion  of  the 

national  laboratories.  However,  the  antigen  used  will  affect  the 

level  at  which  a  serum  is  considered  positive:  -  for  4  HAU  a 

positive  serum  is any  showing  a  titre of  24 or  greater,  for  8 HAU 

a positive serum  is any showing a titre of 23 or greater. 

CHAPTER  5;  Haemagglutination  (HA) test 

Reagents 

1.  Isotonic saline buffered with phosphate  (0.05M) to pH 7.0-7.4. 

2.  Red  blood  cells  (RBC)  taken  and  pooled  from  a  minimum  of  three 

specific  pathogen  free  chickens  (if  not  available  blood  may  be 

taken  from  birds  regularly monitored  and  shown  to be  free of  Avian 

Influenza  antibodies)  into  an  equal  volume  of  Alsever's  solution. 

Cells should  be washed  three  times  in PBS before use. For  the test 

a  1% suspension  (packed  cell  v/v)  is PBS  is recommended. 

3.  The  Community  Reference  Laboratory  will  supply  or  recommend  H5 and 

H7 viruses of  low virulence  for use as standard  antigens. 

Procedure 

1.  Dispensed  0.025ml  PBS  into each well  of  a plastic microtitre  plate 

(V-bottomed  wells should  be used). 

2.  Place  0.025ml  of  virus  suspension  (ie.  allantoic  fluid)  in  the 

first we 11. 

3.  Use  a  microtitration  diluter  to  make  two-fold  dilutions  (1:2  to 

1:4096) of virus across  the plate. 

4.  Dispense a further  0.025ml  of PBS to each well. 

5.  Add 0.025ml  of  1% red blood cells to each well. 

-u-

6.  Mix by  tapping gently  and place at 4°C. 

7.  Plates  are  read  30-40  minutes  later  when  Red  Blood  Cells  control 

are settled. Reading  Is done by tilting  the plate and observing  the 

presence  or  absence  of  tear-shaped  streaming  of  the  RBCs.  Wells 

with  no  HA  should  flow  at  the  same  rate  as  the  control  cells  with 

no virus. 

8.  The  HA  titre  is the  highest  dilution  that  causes  agglutination of 

the  RBCs. That  dilution  may  be  regarded  as  containing  one  HA unit 

(HAU). A more accurate method  for determining  the HA titre  is to do 

HA  tests on virus  from  a close  range of  initial  dilutions  ie. 1:3, 

1:4,  1:5,  1:6,  etc.  This  is  recommended  for  the  accurate 

preparation  of  antigen  for  haemagglutination  inhibition  tests 

(Chapter 6 ). 

CHAPTER  6:  Haemaoglutination  Inhibition  (HI) Test 

Reagents 

1.  Phosphate Buffer  Solution  (PBS). 

2.  Virus containing  allantoic  fluid  diluted with  PBS to contain 4 or 8 

HAU per 0.025ml. 

3.  1% chicken RBCs. 

4.  Negative control  chicken  serum. 

5.  Positive control  serum. 

Procedure 

1.  Dispense  0.025ml  PBS  into  all  wells  of  a  plastic  microtitre  plate 

(with V-bottomed  wells). 

2.  Place 0.025ml  of serum  into first well  of plate. 

u-

3.  Use  microtktration  diluter  to  make  two-fold  dilutions  of  serum 

across  plate. 

4.  Add  0.025ml  of  diluted  allantoic  fluid  containing  4  or  8  HAU. 

5.  Mix  by  tapping  and  place  plate  at  4*C  for  a  minimum  of  60  minutes 

or  room  temperature  for  a minimum  of  30 minutes. 

6.  Add  0.025ml  1% RBCs  to  all  wells. 

7.  Mix  by  gentle  tapping  and  place  at  4°C. 

8.  Plates  are  read  after  30-40  minutes  when  control  RBCs  are  settled. 

This  is  done  by  tilting  and  observing  the  presence  or  absence  of 

tear-shaped  streaming  at  the  same  rate  as  control  wells  containing 

RBCs  (0.025ml)  and  PBS  (0.05ml)  only. 

9.  The  HI  titre  is  the  highest  dilution  of  antiserum  causing  complete 

inhibition  of  4 or  8  units  of  virus  (an  HA  titration  to  confirm  the 

presence  of  the  required  HAU  should  be  included  in each  test). 

10.  The  validity  of  the  results  is  dependent  on  obtaining  a  titre  of 

less  than  23  for  4  HAU  or  22  for  8  HAU  with  the  negative 

control  serum  and  a  titre  of  within  one  dilution  of  the  known  titre 

of  the  positive  control  serum. 

CHAPTER  7;  Intravenous  Pathogenicity  Index  M VP I) 

1.  Infective  allantoic  fluid  from  the  lowest  passage  level  available, 

preferably  from  the  initial  isolation  without  any  selection,  is 

diluted  10~1  in sterile  isotonic  saline. 

2.  0.1ml  diluted  virus  is  injected  intravenously  into  each  of  10  six-

week-old  chickens  (specific  pathogen  free  birds  should  be  used). 

3.  Birds  are  examined  at  24  hour  intervals  for  10 days. 

-n-

4.  At  each  observation  each  bird  is  recorded  normal  (0), sick  (1), 

severely  sick  (2) or dead (3). 

5.  Record  results and calculate  index as shown  in this example: 

Clinical  Signs 

Total  Score 

1  2

3

4

5

6

7

89 

10 

Day after  inoculation 

Normal 
Sick 
Severely  s i c k* 
Dead 

10 
0
0 
0 

2
4
2 
2 

0
2
2 
6 

0
0

0
0

0
0

0
0
2 
0 
8  10  10  10  10  10  10 

00 
00 

0
0

0 

0 

0 

0 

0 

12 

x O -0 
6 x 1 -6 
6 x 2- 
76  X  3  -  228 

12 

TOTAL  " -  246 

Index - mean score per bird per observation - 246 « 2.46 

100 

This  has  to  be  a  subjective  clinical  judgement  but  normally  this 
would  involve  birds  showing  more  than  one  of  the  following  signs: 
respiratory  involvement, depression, diarrhoea, cyanosis of exposed 
skin or wattles, oedema of  face and/or  head, nervous signs. 

CHAPTER  8:  Assessment  of Plague-forming Ability 

1.  It  is usually  best  to use  a dilution  range of  virus  to ensure  that 

an  optimum  number  of  plaques  are  present  on  the  plate.  Ten-fold 

dilutions up  to  10~7  in PBS should  be sufficient. 

2.  Confluent  monolayers of  chick  embryo  cells or  a suitable  cell  line 

(Madin-Darby  bovine  kidney  for  example)  are  prepared  in  5cm 

diameter  Petri  dishes. 

3.  0.2ml  of  each  virus  dilution  is added  to each  of  two  Petri  dishes 

and  the virus allowed  to absorb  for 30 minutes. 

4.  After  washing  three  times with  PBS  the  infected  cells  are overlaid 

with  the  relevant  medium  containing  1%  w/v  agar  and  either 

0.01mg/ml  trypsin  or  no  trypsin.  It  is  important  that  no  serum  is 

added  to be overlay medium. 

-IP-

5.  After  72  hours  incubation  at  37°C  the  plaques  should  be  of 

sufficient  size.  They  are  best  seen  by  removing  the  agar  overlay 

and  staining  the  cell  monolayer  with  crystal  violet  (0.5%  w/v)  in 

25%  v/v  ethanol. 

6.  All  viruses  should  give  clear  plaques  when  incubated  in  the 

presence  of  trypsin  in  the overlay.  When  trypsin  is absent  from  the 

overlay  only  viruses  virulent  for  chickens  will  produce  plaques. 

CHAPTER 9;  Immunodoublediffusion 

The  preferred  method  to  show  the  presence  of  influenza  A  virus  is  to 

demonstrate  the  possession  of  the  nucleocapsid  or  matrix  antigens  which 

are  shared  by  all  influenza  A  viruses.  This  is  generally  done  in 

immunodoublediffusion 

tests 

involving 

either 

concentrated 

virus 

preparations  or  extracts  from  infected  chorioallantoic  membranes. 

Suitable  preparations  of  concentrated  virus  may  be  made  by  simple  high 

speed  centrifugation  of  infectious  allantoic  fluid  and  disruption  of 

virus  to  release  the  internal  nucleocapsid  and  matrix  antigens  by 

treatment 

with 

the  detergent 

sodium 

lauroyl 

sarcosinate. 

Acid 

precipitation  may  also  be  used  by  adding  1N  HCL  to  infective  allantoic 

fluid  to  give  a  final  pH  of  3.5-4.0,  chilling  for  at  least  one  hour  at 

0°C  and  low  speed  centrifugation  at  1000g  for  10  minutes.  The 

supernatant  may  be  discarded  and  the  virus-containing  precipitate 

resuspended  in  a  minimum  volume  of  glycine-sarkosyI  buffer  (1%  sodium 

lauroyl  sarcosinate  buffered  to  pH  9.0  with  a  0.5M  glycine).  These 

preparations  possess  both  nucleocapsid  and  matrix  antigens. 

Beard  (1970)  described  the  preparation  of  nucIeocapsid-rich  antigen 

from  chorioallantoic  membranes  removed  from  infected  eggs.  This  method 

involves:  removal  of  the  chorioallantoic  membranes  from  infected 

haemagglutInln  positive  eggs,  grinding  or  homogenising  the  membranes, 

freezing  and  thawing  three  times  followed  by  centrifugation  at  1000g 

for  10  minutes.  The  pellet  is  discarded  and  the  supernatant  treated 

with  0.1%  formalin  for  use  as  antigen. 

- 2 5-

Either  of  these  two  antigens may  be used  in  immunodoublediffusion  tests 

using  1% agarose,  or  agar,  gels  containing  8.0%  sodium  chloride  made  up 

to  0.1M  phosphate  buffer  pH  7.2.  Influenza  A  virus  is  confirmed  by 

precipitin  lines  formed  by  test  antigen  and  known  positive  antigen 

against  a  known  positive  antiserum  coalescing  to  give  a  line  of 

identity. 

-ÎO-

ANNEX  H 

LIST OF  NATIONAL  AVIAN  INFLUENZA  LABORATORIES 

-  Belgium 

-  Denmark 

-  Germany 

France 

-  Greece 

-  Ireland 

-  Italy 

-  Luxembourg 

-  Netherlands 

-  Portugal 

-  Spain 

Institut  National  de  Recherches 
Vétérinaires,  Groeselenberg  99, 
B  1180  Bruxelles 

National  Veterinary  Laboratory, 
Poultry  Disease  Division,  HangcJvej  2 
DK  8200  Aarhus  N. 

Institut  fur  Kleintierzucht  der 
Bundesforschungsanstalt  fur  Land-
wi rtschaft,  Braunschweig-Vôikenrode, 
Postfach  280,  D  3100  Celle 

Centre  National  d'Etudes  Vétérinaires 
et  Alimentation  -  Laboratoire  Central 
de  Recherches  Agricoles  et  Porcines, 
B.P.  53,  F 22440  Ploufragan 

Veterinary  Research  Laboratory, 
Abbotstown,  Castleknock,  Dublin  15 

Istituto  Patolgie  Aviaire,  Facolta 
di  Medicina  Veterinaria,  Universita 
di  Napoli,  Via  Aniezzo  Falcone  394, 
I 80127  Napoli  F Delpino 1 

Institut  National  de  Recherches 
Vétérinaires,  Groeselenberg  99, 
B  1180  Bruxelles 

Centraal  Diergeneeskundig  Instituut, 
Vestiging  Virologie,  Hourtibweg  39, 
NL  8221  RA  Lelystad 

Laboratôrio  Nactional  de 
Investigaçao  Veterinaria  (LNIV), 
Estrada  de  Benfica  701,  1500  Lisboa 

-  United  Kingdom 

Central  Veterinary  Laboratory,  New 
Haw,  Weybridge,  GB-Surrey  KT15  3NB 

ANNEX  111 

NAME OF THE COMMUNITY  REFERENCE  LABORATORY  FOR AVIAN  INFLUENZA 

Central  Veterinary  Laboratory 

New Haw 

Weybridge 

Surrey KT15 3NB 

UNITED KINGDOM 

ISSN 0254-1475 

COM(91) 304 final 

DOCUMENTS 

EN 

03 

Catalogue number : CB-CO-91-344-EN-C 

ISBN 92-77-74817-6 

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