1992L0040 — EN — 01.01.1995 — 001.001 — 1
This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents
►B COUNCILDIRECTIVE92/40/EEC
of19May1992
introducingCommunitymeasuresforthecontrolofavianinfluenza
(OJ L 167, 22.6.1992, p. 1)
Amended by:
Official Journal
No page date
►A1Act of Accession of Austria, Sweden and Finland C241 21 29.8.1994
(adapted by Council Decision 95/1/EC, Euratom, ECSC) L 1 1 1.1.1995
▼B
COUNCILDIRECTIVE92/40/EEC
of19May1992
introducingCommunitymeasuresforthecontrolofavianinfluenza
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(1),
Having regard to the opinion of the European Parliament(2),
Having regard to the opinion of the Economic and Social
Committee(3),
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 pathogenicform of avian influenza, caused by an influenza virus with specificcharacteristics, and hereinafter termed avian influenza, in order toensure 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 toreduce sharply the profitability of farming or poultry as a whole;
Whereas action must be taken as soon as the presence of the disease is
suspected so that immediate and effective control measures can beimplemented 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 responsible national laboratories, the coordination of whichmust be ensured by the Community reference laboratory;
Whereas common measures for the control of avian influenza form a
basis for maintaining a unified standard with relation to animal health;
Whereas Article 3 of Council Decision 90/424/EEC of 26 June 1990 on
expenditure in the veterinary field(
4) applies in the event of the occur-
rence of avian influenza;
Whereas it is appropriate to confer upon the Commission the task of
taking the necessary applicatory measures,
HAS ADOPTED THIS DIRECTIVE:
Article 1
This Directive defines the Community control measures to be applied
in the event of an outbreak of avian influenza in poultry without preju-dice to the Community provisions governing intra-Community trade.
This Directive shall not apply where avian influenza is detected in
other birds; however, in this case, the Member State concerned shallinform the Commission of any measure it takes.1992L0040 — EN — 01.01.1995 — 001.001 — 2
(1) OJ No C 231, 5. 9. 1991, p. 4.
(2) OJ No C 326, 16. 12. 1991, p. 242.
(3) OJ No C 79, 30. 3. 1992, p. 8.
(4) OJ No L 224, 18. 8. 1990, p. 19. Decision amended by Decision 91/133/
EEC (OJ No L 66, 13. 3. 1991, p. 81).
▼B
Article 2
For the purpose of this Directive, the definitions given in Article 2 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) shall apply as appropriate.
The following definitions shall also apply:
(a)infected poultry shall mean any poultry:
— in which the presence of avian influenza, within the meaning of
Annex I, has been officially confirmed following an examina-tion by an approved laboratory, or
— in the case of second and subsequent outbreaks, in which clin-
ical signs or post-mortem lesions consistent with avianinfluenza are present;
(b)poultry suspected of being infected shall mean any poultry showing
clinical signs or post-mortem lesions which are such that thepresence of avian influenza may reasonably be suspected or anypoultry in which the presence of influenza A virus of subtype H5or H7 has been demonstrated;
(c)poultry suspected of being contaminated shall mean any poultry
which may have been directly or indirectly exposed to the avianinfluenza virus, or influenza A virus of H5 subtype or H7 subtype;
(d)competent authority shall mean the competent authority within the
meaning of Article 2 (6) of Directive 90/425/EEC(
2);
(e)official veterinarian shall mean the veterinarian designated by the
competent authority.
Article 3
Member States shall ensure that there is compulsory and immediate
notification of the suspected presence of avian influenza to the compe-tent authority.
Article 4
1. When poultry in a holding are suspected of being infected or
contaminated with avian influenza, Member States shall ensure that
the official veterinarian immediately activates official investigationarrangements to confirm or rule out the presence of the disease and,in particular, must take or have taken the samples necessary for labora-tory 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 poultrywhich have died, which show clinical signs, and which show nosigns. The record shall be kept up-to-date to include birds born or
dying during the period in which there is a suspicion. The data in
the record shall be kept up-to-date and be produced on request, andmay be checked at each visit;
(b) all poultry on the holding are kept in their living quarters or
confined in some other place where they can be isolated and
without contact with other poultry;
(c) no poultry enter or leave the holding;
(d) all movement
— of persons, other animals and vehicles to or from the holding,1992L0040 — EN — 01.01.1995 — 001.001 — 3
(1) OJ No L 303, 31. 10. 1990, p. 6. Directive as last amended by Directive 91/
496/EEC (OJ No L 268, 24. 9. 1991, p. 56).
(2) Council Directive 90/425/EEC of 26 June 1990 (OJ No L 224, 18. 8. 1990,
p. 29); last amended by Directive 91/496/EEC (OJ No L 268, 24. 9. 1991, p.
56).
▼B
— of poultry meat or carcases, or of animal feed, implements,
waste, droppings, manure litter or anything liable to transmitavian influenza be subject to authorization by the competent
authority;
(e) eggs shall leave the holding with the exception of eggs sent directly
to an establishment approved for the manufacture and/or processingof egg products under Article 6 (1) of Directive 89/437/EEC(
1),
and transported under an authorization which has been granted by
the competent authority. Such authorization must meet the require-
ments laid down in Annex I;
(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 5
are enforced, the owner or keeper of any poultry in which disease is
suspected shall take all reasonable action to ensure compliance withparagraph 2, except for (g) thereof.
4. The competent authority may apply any of the measures provided
for in paragraph 2 to other holdings should their location, their config-uration or contacts with the holding where the disesase is suspectedgive reason to suspect possible contamination.
5. The measures referred to in paragraphs 1 and 2 shall not be with-
drawn until the suspicion of avian influenza has been ruled out by theofficial veterinarian.
Article 5
1. Once the presence of avian influenza has been officially
confirmed on a holding, the Member States shall ensure that thecompetent authority requires, in addition to the measures listed inArticle 4 (2), 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 been killed and all eggs shall be
destroyed. These operations shall be carried out in a way which
minimizes the risk of spreading disease;
(b) any substance or waste, such as animal feed, litter or manures
liable to be contaminated, shall be destroyed or treated appropri-ately. This treatment, carried out in accordance with theinstructions of the official veterinarian, shall ensure the destruction
of any avian influenza virus present;
(c) where poultry from the holding have been slaughtered during the
presumed incubation period of disease the meat from those poultryshall 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 beplaced under official surveillance; table eggs laid during thepresumed incubation period which have been moved from theholding shall whereever possible be traced and destroyed, unlessthey have previously been properly disinfected;
(e) after carrying out operations listed in subparagraphs (a) and (b) the
buildings used for housing poultry, their surroundings, the vehiclesused for transport and all equipment likely to be contaminated shallbe cleaned and disinfescted in accordance with the provisions ofArticle 11;
(f) no poultry shall be reintroduced to the holding until at least 21
days after completion of operations provided for in subparagraph(e);1992L0040 — EN — 01.01.1995 — 001.001 — 4
(1) OJ No L 212, 22. 7. 1989, p. 87. Directive as amended by Directive 89/662/
EEC (OJ No L 395, 30. 12. 1989, p. 13).
▼B
(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 neighbouring holdings should their location, theirconfiguration, or contact with the holding where the disease has beenconfirmed give reason to suspect possible contamination.
Article 6
In the case of holdings which consist of two or more separate flocks,
the competent authority may, in accordance with criteria set by theCommission under the procedure laid down in Article 21, grant a dero-gation from the requirements of Article 5 (1), 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 flocksare completely separate as regards housing, keeping and feeding, sothat the virus cannot spread from one flock to another.
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 whichmay have become infected or contaminated from the same source,
— the movement of persons, poultry or other animals, vehicles, eggs,
meat and carcases 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 thepurpose of carrying out the epidemiological inquiry, a crisis unit shall
be established.
The general rules concerning national crisis units and Community crisis
units will be laid down by the Council, acting by a qualified majorityproposal from the Commission.
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 move-ment of persons, animals or vehicles or in any other way, that holdingshall 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 theirmovements and, where appropriate, to take the action provided for inparagraph 3.
3. When a holding is subject to the official control under paragraphs
1 and 2, the competent authority shall prohibit removal of poultry fromthe holding other than for transport directly to a slaughterhouse underofficial supervision for the purpose of immediate slaughter. Beforegranting 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 movement restrictions referred toin this Article shall be imposed for a period of 21 days from the latestdate of potential contamination; however, such restrictions must applyfor a period of at least seven days.
4. Where it considers that conditions permit, the competent authority
may limit the measures provided for in this Article to a part of theholding and to the poultry contained therein, provided that the poultrythere have been housed, kept and fed completely separately by separatestaff.1992L0040 — EN — 01.01.1995 — 001.001 — 5
▼B
Article 9
1. Once the diagnosis of avian influenza has been officially
confirmed, the Member States shall ensure that the competent authorityestablishes around the infected holding a protection zone based on a
minimum radius of three kilometres, itself contained in a surveillance
zone based on a minimum radius of 10 kilometres. The establishmentof the zones must take account of geographical, administrative, ecolo-gical and epizootiological factors relating to avian influenza, and ofmonitoring facilities.
2. The measures applied in the protection zone shall include:
(a) the identification of all holdings having poultry whithin the zone;
(b) periodic visits to all the holdings having poultry, a clinical exami-
nation of those poultry including, if necessary, the collection ofsamples for laboratory examination; a record of visits and findingsmust 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
carcases and eggs and vehicles carrying poultry, carcases and eggswithin the zone; in general, transport of poultry shall be 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 hasauthorized the transport;
(i) of poultry for immediate slaughter to a slaughterhouse prefer-
ably located in the infected area or, if that is not possible, to a
slaughterhouse designated by the competent authority outsidethe infected area. The special health mark provided for inArticle 5 (1) of Directive 91/494/EEC(
1) must be applied to
this poultrymeat;
(ii) of day-old chicks or ready-to-lay pullets to a holding within
the surveillance zone at which there are no other poultry.This holding must be placed under the official control
provided for in Article 8 (2);
(iii) of hatching eggs to a hatchery designated by the competent
authority; before dispatch, eggs and their packing must be
disinfected. Movements allowed in (i), (ii) and (iii) shall bedirectly executed, under official control. They shall be author-ized only after the official veterinarian has carried out a healthinspection of the holding. The means of transport used must becleaned and disinfected before and after use;
(g) a prohibition on removing or spreading used litter or poultry
manure 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 anddisinfection operations on the infected holding in accordance withArticle 11. The protection zone shall thereafter be part of the surveil-lance zone.
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 slaughterhouseoutside the surveillance zone designated by the competent1992L0040 — EN — 01.01.1995 — 001.001 — 6
(1) OJ No L 268, 24. 9. 1991, p. 35.
▼B
authority. The special health mark provided for in Article 3 of
Directive 91/494/EEC must be applied to this poultry meat;
(d) a prohibition on the movement of hatching eggs out of the surveil-
lance zone unless to a hatchery designated by the competent
authority. Before dispatch the eggs and their packing must be disin-fected;
(e) a prohibition on the movement of used litter or poultry manure out
of the zone;
(f) a prohibition of fairs, markets, shows or other gatherings of poultry
and other birds;
(g) without prejudice to the provisions of (a) and (b), the prohibition of
transport of poultry except for transit by major highways or rail-ways.
5. The measures applied in the surveillance zone shall be maintained
for at least 30 days after the carrying out of preliminary cleaning anddisinfection operations on the infected holding in accordance with
Article 11.
6. Where the zones are situated in the territory of more than one
Member State, the competent authorities of the Member States
concerned shall cooperate in establishing the areas described in para-graph 1. However, if necessary, the protection zone and thesurveillance zone shall be established by the procedure provided for inArticle 21.
Article 10
Member States shall ensure that:
(a) the competent authority determines the arrangements allowing them
to trace the movement of eggs and poultry;
(b) the owner or keeper of poultry is required to supply the competent
authority, in response to any request by that authority, with infor-mation concerning poultry and eggs entering or leaving hisholding;
(c) all persons engaged in the transport or marketing of poultry and
eggs are able to supply the competent authority with information
concerning the movements of poultry and eggs which they havetransported or marketed and to furnish all the details concerningsuch information.
Article 11
Member States shall ensure that:
(a) the disinfectants to be used and their concentrations are officially
approved by the competent authority;
(b) the cleaning and disinfection operations are carried out under offi-
cial supervision, in accordance with:
(i) instructions given by the official veterinarian,
(ii) the procedure for cleaning and disinfecting an infected holding,
as laid down in Annex II.
Article 12
Collection of samples and laboratory testing to detect the presence of
avian influenza virus shall be carried out in accordance with Annex III.
Article 13
Member States shall ensure that the competent authority takes all the
necessary measures for persons established in the protection andsurveillance zones to be informed of the restrictions in force andmake all necessary arrangements for the appropriate implementationof the measures in question.1992L0040 — EN — 01.01.1995 — 001.001 — 7
▼B
Article 14
1. Member States shall ensure that, in each Member State there is
designated:
(a) a national laboratory at which facilities and expert personnel shall
be maintained to permit assessment of the pathogenicity of influ-enza virus isolates, in accordance with Annex III, Chapter 7, andidentification of influenza A viruses of H5 or H7 subtypes;
(b) a national laboratory at which reagents for use in regional labora-
tories are tested;
(c) a national institute or laboratory 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 IV shall be responsible
for coordinating standards and methods of diagnosis, use of reagents
and testing of vaccines.
3. The national laboratories listed in Annex IV shall be responsible
for coordinating the standards and diagnostic methods laid down ineach avian influenza diagnostic laboratory within the Member State.
To this end:
(a) they may provide diagnostic reagents to national 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 IV shall liaise with the
Community reference laboratory referred to in Article 15.
Article 15
The Community reference laboratory for avian influenza is mentioned
in Annex V. Without prejudice to the provisions of Decision 90/424/EEC, and in particular Article 28 thereof, the powers and duties ofthe laboratory shall be those appearing in the said Annex.
Article 16
Vaccination against avian influenza with vaccines authorized by the
competent authority may only be used to supplement the controlmeasures carried out when the disease appears and in accordance with
the following provisions:
(a) 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 proce-
dure laid down in Article 21. This decision shall have particularregard 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.
However, by way of derogation from the first subparagraph, the
decision to introduce emergency vaccination around the outbreakmay be taken by the Member State concerned, following notifica-tion to the Commission, provided the fundamental interests of theCommunity are not jeopardized. Such decision will be re-examined1992L0040 — EN — 01.01.1995 — 001.001 — 8
▼B
immediately within the Standing Veterinary Committee in accor-
dance with the procedure provided for in Article 21;
(b) where a Member State is authorized, in accordance with point (a),
to have recourse to emergency vaccination on a limited part of itsterritory the status of the remainder of the territory shall not beaffected, provided that the immobilization measures for the vacci-
nated animals are effective during a period to be determined 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 ofavian influenza.
This plan must allow access to facilities, equipment, personnel and all
other appropriate materials necessary for the rapid and efficient eradi-cation of the outbreak.
2. The criteria to be applied for drawing up the plan are laid down
in Annex VI.
3. Plans drawn upin accordance with the criteria listed in Annex VI
shall be submitted to the Commission not later than six months afterthis Directive is brought into application.
4. The Commission shall examine the plans in order to determine
whether they permit the desired objective to be attained and shallsuggest 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 accor-
dance with the same procedure, to take into account developments in
the situation.
Article 18
1. Commission experts may, in collaboration with the competent
authorities, and insofar as is necessary to ensure uniform applicationof this Directive, make on-the-spot checks. In order to do this, they
may check a representative percentage of establishments to see whether
the competent authorities are checking that these establishments arefulfilling the requirements of this Directive. The Commission shallinform the Member States of the result of the checks carried out.
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 deter-
mined in accordance with the procedure laid down in Article 21.
Article 19
The detailed conditions governing the Community's financial contribu-
tion to the measures connected with the application of this Directive
are laid down in Decision 90/424/EEC.
Article 20
The Annexes shall be amended, as and when required, by the Council
acting by a qualified majority acting on a proposal from the Commis-sion in particular in order to take into account developments inresearch and in diagnostic procedures.1992L0040 — EN — 01.01.1995 — 001.001 — 9
▼B
Article 21
1. Where the procedure laid down in this Article is to be followed,
the Standing Veterinary Committee, set upby Decision 68/361/EEC(1),
hereinafter referred to as the ‘the Committee’, shall be informed
without delay by its Chairman either on his own initiative or at therequest of the representative of a Member State.
2. The representative of the Commission shall submit to the
committee a draft within a time limit which the chairman may laydown according to the urgency of the matter. The opinion shall bedelivered by the majority laid down in Article 148 (2) of the Treaty
in the case of decisions which the Council is required to adopt on a
proposal from the Commission. The votes of the representatives of theMember States within the committee shall be weighted in the mannerset out in that Article. The chairman shall not vote.
3. (a) The Commission shall adopt the measures envisaged if they are
in accordance with the opinion of the committee.
(b) If the measures envisaged are not in accordance with the opinion
of the committee, or if no opinion is delivered, the Commissionshall, without delay, submit to the Council a proposal relating tothe measures to be taken. The Council shall act by a qualifiedmajority.
If, on the expiry of a period of three months from the date of
referral to the Council, the Council has not acted, the proposedmeasures shall be adopted by the Commission save where theCouncil has decided against the said measures by a simple
majority.
Article 22
Member States shall bring into force the laws, regulations and admin-
istrative provisions necessary to comply with this Directive before 1
January 1993. They shall forthwith inform the Commission thereof.
When Member States adopt these measures, they shall contain a refer-
ence to this Directive or shall be accompanied by such reference on theoccasion of their official publication. The methods of making such areference shall be laid down by the Member States.
Article 23
This Directive is addressed to the Member States.1992L0040 — EN — 01.01.1995 — 001.001 — 10
(1) OJ No L 265, 18. 10. 1968, p. 23.
▼B
ANNEX I
AUTHORIZATIONTOREMOVEEGGSFROM AHOLDINGSUBJECT
TOTHECONDITIONSOFARTICLE4(2)(e)OFTHISDIRECTIVE
The authorization issued by the competent authority to transport eggs from a
suspect holding subject to the provisions of Article 4 (2) (e) to an establishment
approved for the manufacture and processing of egg products in accordance with
the provisions of Article 6 (1) of Directive 89/437/EEC, hereinafter called the
designated establishment, must meet the following conditions:
1. in order to be allowed to be removed from a suspect undertaking, eggs must:
(a) comply with the requirements laid down in Chapter IV of the Annex to
Directive 89/437/EEC;
(b) be sent directly from the suspect undertaking to the designated establish-
ment; each consignment must be sealed before dispatch by the official
veterinarian of the suspect holding and must remain sealed throughout
transport to the designated establishment;
2. the official veterinarian of the suspect undertaking shall inform the compe-
tent authority of the designated establishment of this intention of sending
eggs to it;
3. the competent authority responsible for the designated establishment shall
ensure that:
(a) eggs referred to in 1 (b) will be kept isolated from other eggs from the
time they arrive until they are processed;
(b) the shells of such eggs shall be regarded as high-risk material in accor-
dance with Article 2 (2) of Directive 90/667/EEC(1) and shall be dealt
with in accordance with the requirements of Chapter II of that Directive;
(c) the packaging material, the vehicles used to transport eggs referred to in
1 (b) and all premises with which the eggs come into contact are cleaned
and disinfected in such a way as to destroy all avian influenza virus;
(d) the official veterinarian of the suspect holding shall be informed of all
consignments of processed eggs.1992L0040 — EN — 01.01.1995 — 001.001 — 11
(1) OJ No L 363, 27. 12. 1990, p. 51.
▼B
ANNEX II
PROCEDURE FOR CLEANING AND DISINFECTING AN INFECTED
HOLDING
I.Preliminary cleaninganddisinfecting
(a) As soon as the carcases of the poultry have been removed for disposal,
those parts of the premises in which the poultry was housed and any
parts of other buildings, yards etc. contaminated during slaughter or
post-mortem examination should be sprayed with disinfectants approved
for use in accordance with Article 11 of this Directive.
(b) Any tissue of poultry or eggs which could have contaminated buildings,
yards, utensils etc. should be carefully collected and disposed of with the
carcases.
(c) The used disinfectant must remain on the surface for at least 24 hours.
II.Finalcleaninganddisinfection
(a) Grease and dirt should be removed from all surfaces by the application
of a degreasing agent and washed with water,
(b) After washing with water as described in (a), further spraying with disin-
fectant should be applied,
(c) After seven days the premises should be treated with a degreasing agent,
rinsed with cold water, sprayed with disinfectant and rinsed again with
water.
(d) Used litter and manure must be treated by a method capable of killing
the virus. This method must comprise one of the following practices:
(i) incineration or steam treatment at a temperature of 70º C;
(ii) burying deepenough to p revent access by vermin and wild birds;
(iii) stacking and dampening (if necessary to facilitate fermentation),
covering to keepin the heat so that a temp erature of 20º C is
attained and leaving covered for 42 days so as to prevent access
by vermin and wild birds.1992L0040 — EN — 01.01.1995 — 001.001 — 12
▼B
ANNEX III
DIAGNOSTIC PROCEDURES FOR THE CONFIRMATION AND
DIFFERENTIALDIAGNOSISOFAVIANINFLUENZA(AI)
The following procedures for the isolation and characterization of avian influ-
enza 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 differen-
tial 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 I
Samplingandtreatmentofsamples
1.Samples
Cloacal 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 in paragraph 1 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 homogenized (in an enclosed blender or using a pestle and mortar
and sterile sand) in antibiotic medium and made to 10—20 % w/v suspen-
sions in the medium. The suspensions should be left for about two hours at
ambient temperature (or longer periods at 4º C) and then clarified by centri-
fugation (e.g. 800 to 1000 × g for 10 minutes).
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
samples and a typical mixture is: 10000 units/ml penicillin, 10 mg/ml strep-
tomycin, 0,25 mg/ml gentamycin and 5 000 units/ml mycostatin in phosphate
buffered saline. These levels can be reduced upto five-fold for tissues and
tracheal swabs. For control of Chlamydia organisms 50 mg/ml oxytetracy-cline 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
Virusisolation
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?A eggs
which have been incubated for eight to 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 37º 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 4º C and the allantoic-amniotic fluids
tested for haemagglutination 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 450 nm
membrane filter, further antibiotics added and inoculated into embryonated
eggs as above.1992L0040 — EN — 01.01.1995 — 001.001 — 13
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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 inhibi-
tion test as described in Chapters 5 and 6. Positive inhibition i.e. 24,o r
more, with polyclonal antisera specific for H5 or H7 subtypes of influenza
A and of a titre of at least 29would serve as preliminary identification
enabling the imposition of interim control measures.
2.Confirmatory identification
Since there are 13 haemagglutinin subtypes and nine neuraminidase subtypes
of influenza viruses and variations occur within each of these it is not prac-
ticable 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 immunodouble-
diffusion test to detect groupantigens as described in Chap ter 9
(immunofluorescence or ELISA techniques to detect groupantigens
may be used if preferred by the national laboratory);
(ii) determine whether or not the isolate is of H5 or H7 subtype;
(iii) carry out an intravenous pathogenicity index test in six-week-old
chickens as described in Chapter 7. Intravenous pathogenicity indices
of greater than 1.2 indicate the presence of virus requiring a full imple-
mentation 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 charac-
terization.
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 epizootiology 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 amino acids at the cleavage site of the
haemagglutinin protein should also be carried out.
CHAPTER 4
Seriological testsforavianinfluenzavirusantibodies
1. 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 haemagglutination
inhibition tests as described in Chapters 5 and 6.
If the haemagglutinin subtype is not known, evidence for infection with
influenza A viruses may be obtained by detecting antibodies directed to the
groupsp ecific antigens.
For this purpose either an immunodoublediffusion test (as described in
Chapter 9) 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 immunodouble-
diffusion 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.
2. (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 % probability1992L0040 — EN — 01.01.1995 — 001.001 — 14
▼B
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 haemagglutination
inhibition tests as defined in Chapter 6.
There is some debate as to whether 4 or 8 haemagglutinin units should
be used for the H1 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 24or greater, for 8 HAU a positive serum is any showing a titre
of 23or 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 regu-
larly monitored and shown to be free of Avian influenza antibodies) into an
equal volume of Alsever?As solution. Cells should be washed three times in
PBS before use. For the other test a 1 % suspension (packed cell v/v) in 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. Dispense 0,025 ml PBS into each well of a plastic microtitre plate (V-
bottomed wells should be used).
2. Place 0,025 ml of virus suspension (i.e. allantoic fluid) in the first well.3. Use a microtitration diluter to make two-fold dilutions (1:2 to 1:4 096) of
virus across the plate.
4. Dispense a further 0,025 ml of PBS to each well.5. Add 0,025 ml of 1 % red blood cells to each well.6. Mix by tapping gently and place at 4º 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 i.e. 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
Haemagglutinationinhibition(Hl)test
Reagents
1. Phosphate buffer solution (PBS).2. Virus containing allantoic fluid diluted with PBS to contain 4 or 8 HAU per
0,025 ml.
3. 1 % chicken RBCs.4. Negative control chicken serum.
5. Positive control serum.1992L0040 — EN — 01.01.1995 — 001.001 — 15
▼B
Procedure
1. Dispense 0,025 ml PBS into all wells of a plastic microtitre plate (with V-
bottomed wells).
2. Place 0,025 ml of serum into first well of plate.
3. Use microtitration diluter to make two-fold dilutions of serum across plate.
4. Add 0,025 ml of diluted allantoic fluid containing 4 or 8 HAU.5. Mix by tapping and place plate at 4º C for a minimum of 60 minutes or
room temperature for a minimum of 30 minutes.
6. Add 0,025 ml 1 % RBCs to all wells.
7. Mix by gentle tapping and place at 4º 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 (0,025 ml and
PBS (0,05 ml) only.
9. The Hl titre is the highest dilution of antiserum causing complete inhibition
of four or eight 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 2
3
for 4 HAU or 22for 8 HAU with a negative control serum and a titre of
within one dilution of the known titre of the positive control serum.
CHAPTER 7
IntravenousPathogenicityIndex(IVPI)
1. Infective allantoic fluid from the lowest passage level available, preferably
from the initial isolation without any selection, is diluted 101in sterile
isotonic saline.
2. 0,1 ml 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.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 SignsDay after inoculation
Total Score
123456789 1 0
Normal 10 2 0 0 0 0 0 0 0 0 12 × 0 = 0
Sick 0 4 2 0 0 0 0 0 0 0 6 × 1 = 6
Severely sick (*) 0 2 2 2 0 0 0 0 0 0 6 × 2 = 12
Dead 0 2 6 8 10 10 10 10 10 10 76 × 3 = 228
Total = 246
Index¼mean score per bird per observation ¼246
100¼2;46
(*) This has to be a subjective clinical judgment 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
Assessmentofplaque-formingability
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 10−7in
PBS should be sufficient.1992L0040 — EN — 01.01.1995 — 001.001 — 16
▼B
2. Confluent monolayers of chick embryo cells or a suitable cell line (Madin-
Darby bovine kidney for example) are prepared in 5 cm diameter Petri
dishes.
3. 0,2 ml 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 0,01 mg/ml trypsin or
no trypsin. It is important that no serum is added to the overlay medium.
5. After 72 hours incubation at 37º C the plaques should be of sufficient size.
They are best seen by removing the agar overlay and staining the cell mono-
layer 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 demon-
strate the possession of the nucleocapsid or matrix antigens which are shared
by all influenza A virus. This is generally done in immunodoublediffusion tests
involving either concentrated virus preparations or extracts from infected chor-
ioallantoic 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 precipatation 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 0º C and low speed centrifugation at 1 000 g for 10 minutes.
The supernatant may be discarded and the virus-containing precipitate resus-
pended in a minimum volume of glycine-sarkosyl 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 nucleocapsid-rich antigen from chor-
ioallantoic membranes removed from infected eggs. This method involves:
removal of the chorioallantoic membranes from infected haemagglutinin posi-
tive eggs, grinding or homogenising the membranes, freezing and thawing
three times followed by centrifugation at 1 000 g for 10 minutes. The pellet is
discarded and the supernatant treated with 0,1 % formalin for use as antigen.
Either of these two antigens may be used in immunodoublediffusion tests using
1 % agarose, or agar, gels containing 8,0 % sodium chloride made upto 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.1992L0040 — EN — 01.01.1995 — 001.001 — 17
▼B
ANNEX IV
LISTOFNATIONALAVIANINFLUENZALABORATORIES
Belgium Institut National de Recherches Vétérinaires,
Groeselenberg 99, B-1180 Brussels
Denmark National Veterinary Laboratory, Poultry Disease Division,
Hangøvej 2, DK-8200 Aarhus N
Germany Institut für Kleintierzucht der Bundesforschungsanstalt für
Landwirtschaft,
Braunschweig-Völkenrode, Postfach 280, D-3100 Celle
France Centre National d'Etudes Vétérinaires et Alimentaires —
Laboratoire Central de Recherches Avicoles et Porcines,
B.P. 53, F-22440 Ploufragan
Greece Ινστιτούτο Λοιµωδών και Παρασιτικών Νοσηµάτων
66, 26 ης Οκτωβρίου, GR-54627 Θεσσαλονίκη
Institute of Infections and Parasitological Diseases,
66, 26th October Street, 546 27 Thessaloniki
Ireland Veterinary Research Laboratory,
Abbotstown, Castleknock, Dublin 15
Italy Istituto Patologie Aviaire, Facoltà di Medicina Veterinaria,
Università di Napoli,
via Aniezzo, Falcone 394, I-80127 Napoli F Delpino 1
Luxembourg Institut National de Recherches Vétérinaires,
Groeselenberg 99, B 1180 Brussels
Netherlands Centraal Diergeneeskundig Instituut, Vestiging Virologie,
Houtribweg 39, NL-8221 RA Lelystad
Portugal Laboratório Nacional de Investigação Veterinária (LNIV),
Estrada de Benfica 701, P-1500 Lisbon
Spain Centro Nacional de Referencia para la Peste Aviar es el Labor-
atorio Nacional de Sanidad y Producción Animal de Barcelona,
Zona Franca Circunvalación-Tramo 6, Esquina Calle 3, Barce-
lona
United Kingdom Central Veterinary Laboratory,
New Haw, UK-Weybridge, Surrey KT15 3NB
Austria Bundesanstalt für Virusseuchenbekämpfung bei Haustieren,
Wien-Hetzendorf
Finland Eläinlääkintä- ja elintarvikelaitos, Helsinki — Anstalten för
veterinärmedicin och livsmedel, Helsingfors
Sweden Statens veterinärmedicinska anstalt, Uppsala1992L0040 — EN — 01.01.1995 — 001.001 — 18
▼A1
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ANNEX V
COMMUNITYREFERENCELABORATORYFORAVIANINFLUENZA
Name of Laboratory
Central Veterinary Laboratory,
New Haw,
UK-Weybridge,
Surrey KT 15 3NB,
United Kingdom.
The functions and duties of the EC reference laboratory for avian influenza shall
be:
1. to coordinate, in consultation with the EC Commission, the methods
employed in the Member States for diagnosing avian influenza. Specifically
by:
(a) typing, storing and supplying strains of avian influenza virus for serolo-
gical tests and the preparation of antisera;
(b) supplying standard sera and other reference reagents to the National
Reference Laboratories in order to standardize the tests and reagents
used in the Member States;
(c) building upand retaining a collection of avian influenza virus strains and
isolates;
(d) organizing periodical comparative tests of diagnostic procedures at
Community level;
(e) collecting and collating data and information on the methods of diagnosis
used and the results of tests carried out in the Community;
(f) characterizing isolates of avian influenza viruses by the most up-to-date
methods available to allow greater understanding of the epizootiology of
avian influenza and to gain an insight into the epizootiology of the virus
and the emergence of highly pathogenic and potentially pathogenic
strains;
(g) keeping abreast of developments in avian influenza surveillance, epizoo-
tiology and prevention throughout the world;
(h) retaining expertise on avian influenza virus and other pertinent viruses to
enable rapid differential diagnosis;
(i) acquiring a thorough knowledge of the preparation and use of the
products of veterinary immunology used to eradicate and control avian
influenza;
2. to actively assist in the diagnosis of avian influenza outbreaks in Member
States by receiving virus isolates for confirmatory diagnosis, characterization
and epizootiological studies. In particular, the laboratory should be able to
carry out nucleotide sequencing analysis to allow determination of the
deduced amino acid sequence at the cleavage site of the haemagglutin mole-
cule of avian influenza viruses of H5 or H7 subtpye,
3. to facilitate the training or retraining of experts in laboratory diagnosis with a
view to the harmonization of techniques throughout the Community.1992L0040 — EN — 01.01.1995 — 001.001 — 19
▼B
ANNEX VI
CRITERIAFORCONTINGENCYPLANS
Contingency plans shall meet at least the following criteria:
1. the establishment of a crisis centre on a national level, which shall coordi-
nate all control measures in the Member State concerned;
2. a list shall be provided of local disease control centres with adequate facil-
ities to coordinate the disease control measures at a local level;
3. detailed information shall be given about the staff involved in control
measures, their skills and their responsibilities;
4. each local disease control centre must be able to contact rapidly persons/
organizations which are directly or indirectly involved in an outbreak;
5. equipment and materials shall be available to carry out the disease control
measures properly;
6. detailed instructions shall be provided on action to be taken on suspicion
and confirmation of infection or contamination, including proposed means
of disposal of carcases;
7. training programmes shall be established to maintain and develop skills in
field and administrative procedures;
8. diagnostic laboratories must have facilities for post-mortem examination,
the necessary capacity for serology, histology etc. and must maintain the
skills for rapid diagnosis. Arrangements must be made for rapid transporta-
tion of samples;
9. details shall be provided of the quantity of avian influenza vaccine esti-
mated to be required in the event of a reinstatement of emergency
vaccination;
10. provisions shall be made to ensure the legal powers necessary for the imple-
mentation of the contingency plans.1992L0040 — EN — 01.01.1995 — 001.001 — 20