CELEX: 51991PC0304
Language: en
Date: 1991-07-30
Title: PROPOSAL FOR A COUNCIL REGULATION ( EEC ) INTRODUCING COMMUNITY MEASURES FOR THE CONTROL OF AVIAN INFLUENZA

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)
 ---pagebreak---                                     - 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
 ---pagebreak---                              - 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.
 ---pagebreak---                                          -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;
 ---pagebreak---                                      - 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:
                                   Article 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.
 ---pagebreak---                                        - 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
                                                             (1)
    third   countries of poultry       and  hatching    eggs      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
 ---pagebreak---                                       _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;
 ---pagebreak---                                     - 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;
 ---pagebreak---                                    - 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.
 ---pagebreak---                                           - 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)
 ---pagebreak---                                      - 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.
 ---pagebreak---                                     - 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:
 ---pagebreak---                                    - 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
 ---pagebreak---                                   - 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.
 ---pagebreak---                                       - 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 l e 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;.
 ---pagebreak---                                       - 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
 ---pagebreak---                                    - 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";
 ---pagebreak---                                   - 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.
 ---pagebreak---                                    - 16 -
2.  The criteria   to be applied    for drawing up the contingency         plan
                                                                     (1
    shall be those laid down in Commission Decision 91/42/EEC ) 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
 ---pagebreak---                                     - 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
 ---pagebreak---                                     - 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
 ---pagebreak---                                     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).
 ---pagebreak---                                         - 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.
 ---pagebreak---                                         -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
                        4
   inhibition ie. 2 , or more, with polyclonal antisera specific for
   H5 or H7 subtypes of influenza A and of a titre of at least 2 9
   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;
 ---pagebreak---                                      -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.
 ---pagebreak---                                        -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.
 ---pagebreak---                                          -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
                                                    4
    positive serum is any showing a titre of 2        or greater, for 8 HAU
    a positive serum is any showing a titre of 2 3 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.
 ---pagebreak---                                             -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.
 ---pagebreak---                                                  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
                    3
    less   than   2     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 V P I)
1.  Infective allantoic fluid from the lowest passage level available,
    preferably    from    the   initial   isolation  without  any  selection,  is
                 1
    diluted 10~      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.
 ---pagebreak---                                                -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:
                                 Day after inoculation
 Clinical Signs                                                         Total Score
                       1    2    3   4  5    6    7   8    9      10
 Normal               10   2    0   0   0    0    0   0    0    0       12    x O - 0
 Sick                  0   4    2   0   0   0     0   0    0   0           6 x 1 - 6
 Severely s i c k *    0   2     2    2   0    0    0    0    0    0     6 x 2 -    12
 Dead                  0   2     6    8 10 10 10 10 10 10               76 X 3 - 228
                                                                        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.
 ---pagebreak---                                                 -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.
 ---pagebreak---                                          -25-
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.
 ---pagebreak---                                           -ÎO-
                                ANNEX   H
             LIST OF NATIONAL AVIAN INFLUENZA LABORATORIES
- Belgium                          Institut National de Recherches
                                   Vétérinaires, Groeselenberg 99,
                                   B 1180 Bruxelles
- Denmark                          National Veterinary Laboratory,
                                   Poultry Disease Division, HangcJvej 2
                                   DK 8200 Aarhus N.
- Germany                           Institut fur Kleintierzucht der
                                   Bundesforschungsanstalt fur Land-
                                   wi rtschaft, Braunschweig-Vôikenrode,
                                   Postfach 280, D 3100 Celle
  France                          Centre National d'Etudes Vétérinaires
                                  et Alimentation - Laboratoire Central
                                  de Recherches Agricoles et Porcines,
                                  B.P. 53, F 22440 Ploufragan
- Greece
- Ireland                          Veterinary Research Laboratory,
                                   Abbotstown, Castleknock, Dublin 15
- Italy                            Istituto Patolgie Aviaire, Facolta
                                   di Medicina Veterinaria, Universita
                                   di Napoli, Via Aniezzo Falcone 394,
                                   I 80127 Napoli F Delpino 1
- Luxembourg                        Institut National de Recherches
                                   Vétérinaires, Groeselenberg 99,
                                   B 1180 Bruxelles
- Netherlands                      Centraal Diergeneeskundig Instituut,
                                   Vestiging Virologie, Hourtibweg 39,
                                   NL 8221 RA Lelystad
- Portugal                         Laboratôrio Nactional de
                                   Investigaçao Veterinaria (LNIV),
                                   Estrada de Benfica 701, 1500 Lisboa
- Spain
- United Kingdom                   Central Veterinary Laboratory, New
                                   Haw, Weybridge, GB-Surrey KT15 3NB
 ---pagebreak---                                 ANNEX 111
     NAME OF THE COMMUNITY REFERENCE LABORATORY FOR AVIAN INFLUENZA
Central Veterinary Laboratory
New Haw
Weybridge
Surrey KT15 3NB
UNITED KINGDOM
 ---pagebreak---                                                                      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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