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) 
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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 
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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. 
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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; 
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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. 
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Article 2 
1. For the purpose of this Regulation, the definitions given in 
Article 2 of Council Directive 90/539/EEC on animal health 
conditions governing intra-Community trade in, and imports from 
third countries of poultry and hatching eggs(1) shall apply as 
appropriate. 
2. The following definitions shall also apply: 
(a) "infected poultry" means any poultry: 
in which the presence of avian influenza, in the meaning of 
Annex I, has been officially confirmed following an 
examination by an approved laboratory, or 
in the case of second and subsequent outbreaks in which 
clinical signs or post-mortem lesions consistent with avian 
influenza are present; 
(b) "poultry suspected of being infected" means any poultry showing 
clinical signs or post-mortem lesions which are such that the 
presence of avian influenza may reasonably be suspected; or the 
presence of influenza A virus of subtype H5 or H7 has been 
demonstrated. 
(c) "poultry suspected of being contaminated" means any poultry 
which may have been directly or indirectly exposed to the avian 
influenza virus, or influenza A virus of H5 subtype or H7 
subtype. 
(d) "competent authority": veterinary authority appointed for the 
purpose by the national administration of the country 
concerned, being directly responsible to the national 
administration within the scope of this Regulation, and 
reporting through the national administration. 
(e) "official veterinarian": the veterinarian designated by the 
competent authority of the Member State. 
(1) OJ No L 303, 31.10.1990, p. 6 
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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; 
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(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; 
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(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. 
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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) 
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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. 
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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: 
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(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 
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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. 
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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. 
Article 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;. 
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(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 
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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"; 
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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. 
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2. The criteria to be applied for drawing up the contingency plan 
shall be those laid down in Commission Decision 91/42/EEC(1) of 8 
January 1991 laying down the criteria to be applied when drawing up 
contingency plans for the control of foot and mouth disease, in 
application of Article 5 of Council Directive 90/423/EEC, which 
shall apply mutatis mutandis. 
The Commission may, in accordance with Article 21, amend or 
supplement those criteria taking into account the specific nature 
of avian influenza. 
3. Plans drawn up in accordance with the criteria provided for in 
paragraph 2 shall be submitted to the Commission not later than 12 
months after this regulation enters into force. 
4. The Commission shall examine the plans in order to determine 
whether they permit the desired objective to be attained and shall 
suggest to the Member State concerned any amendments required in 
particular to ensure that they are compatible with those of the 
other Member States. 
The Commission shall approve the plans, if necessary amended, in 
accordance with the procedure laid down in Article 21. 
The plans may subsequently be amended or supplemented, in 
accordance with the same procedure, to take into account 
developments in the situation. 
ArtiCie 18 
Veterinary experts from the Commission may, in collaboration with the 
authorities of the concerned Member State, insofar as is necessary to 
ensure uniform application of this Regulation, make on-the-spot checks-, 
the Commission shall inform the Member States of the results of the 
investigation. 
(1) OJ No L 23, 29.01.1991, p. 29 
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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 
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The Commission shall take the utmost account of the opinion 
delivered by the Committee. It shall inform the Committee of the 
manner in which its opinion has been taken into account. 
Article 22 
This Regulation shall enter into force on 1 July 1991. 
This Regulation shall be binding in its entirety and directly 
applicable in all Member States. 
Done at Brussels For the Council 
ANNEX I 
DIAGNOSTIC PROCEDURES FOR THE CONFIRMATION 
AND DIFFERENTIAL DIAGNOSIS OF AVIAN INFLUENZA (AI) 
The following procedures for the isolation and characterization of 
avian influenza viruses should be regarded as guidelines and the minima 
to be applied In the diagnosis of the disease. 
For the purpose of the diagnostic procedures for the confirmation and 
differential diagnosis of avian influenza the following definition 
shall apply. 
"Avian influenza" means an infection of poultry caused by any influenza 
A virus which has an intravenous pathogenicity index in six-week-old 
chickens greater than 1.2 or any infection with influenza A viruses of 
H5 or H7 subtype for which nucleotide sequencing has demonstrated the 
presence of multiple basic amino acids at the cleavage site of the 
haemagglutinin. 
CHAPTER 1: Sampling and treatment of samples 
1. Samples 
Cioacal swabs (or faeces) and tracheal swabs from sick birds; 
faeces or intestinal contents, brain tissue, trachea, lungs, liver, 
spleen and other obviously affected organs from recently dead 
birds. 
2. Treatment of samples 
The organs and tissues listed above may be pooled, but separate 
treatment of faecal material is essential. Swabs should be placed 
in sufficient antibiotic medium to ensure full immersion. Faeces 
samples and organs should be homogenised (in an enclosed blender or 
using a pestle and mortar and sterile sand) in antibiotic medium 
and made to 10-20% w/v suspensions in the medium. The suspensions 
should be left for about two hours at ambient temperature (or 
longer periods "at 4*C) and then clarified by centrifugation (eg. 
800 to 1000 x g for 10 minutes). 
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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. 
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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 
inhibition test as described in Chapters 5 and 6. Positive 
inhibition ie. 24, or more, with polyclonal antisera specific for 
H5 or H7 subtypes of influenza A and of a titre of at least 29 
would serve as preliminary identification enabling the imposition 
of interim control measures. 
2. confirmatory identification 
Since there are 13 haemagglutinin subtypes and 9 neuraminidase 
subtypes of influenza viruses and variations occur within each of 
these it is not practicable nor cost effective for each national 
laboratory to hold antisera which will allow full antigenic 
characterization of influenza isolates. However, each national 
laboratory should: 
(i) confirm that the isolate is an influenza A virus using an 
immunodoublediffusion test to detect group antigen as 
described in Chapter 9 of this Annex (immunofluorescence or 
EL I SA techniques to detect group antigens may be used if 
preferred by the national laboratory); 
(Ii) determine whether or not the isolate is of H5 or H7 
subtype; 
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(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. 
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CHAPTER 4; Serological tests for avian jnfluenza 
virus antibodies 
During eradication programmes where the H subtype of the virus 
responsible is already known, or by using the homologous virus as 
antigen, serological monitoring for evidence of infection may be 
done using haemaggluniation inhibition tests as described in 
Chapters 5 and 6. 
If the haemagglutinin subtype is not know, evidence for infection 
with influenza A viruses may be obtained by detecting antibodies 
directed to the group specific antigens. For this purpose either an 
immunodoublediffusion test (as described in Chapter 9 of this 
Annex) or an ELISA test may be used (a problem with ELISA is the 
host specificity of the test since it is dependent on the detection 
of host immunoglobulins). Waterfowl rarely give positive results in 
immunodoublediffusion tests and, unless the subtype is known, it is 
probably only practicable to examine such birds for the presence of 
antibodies to H5 and H7 subtypes. 
(a) samples 
Blood samples should be taken from all birds if the flock size 
is less than 20 and from 20 birds from larger flocks (This will 
give a >99% probability of detecting at least one positive 
serum if 25% or more of the flock is positive, regardless of 
flock size). The blood should be allowed to clot and serum 
removed for testing. 
(b) Examination for antibodies 
Individual serum samples should be tested for their ability to 
inhibit influenza virus haemagglutinating antigen in standard 
haemagglut(nation inhibition tests as defined in Chapter 6. 
-Ik-
There is some debate as to whether 4 or 8 haemagglut inin units 
should be used for the HI tests. It would appear that either is 
valid and the choice should be left to the discretion of the 
national laboratories. However, the antigen used will affect the 
level at which a serum is considered positive: - for 4 HAU a 
positive serum is any showing a titre of 24 or greater, for 8 HAU 
a positive serum is any showing a titre of 23 or greater. 
CHAPTER 5; Haemagglutination (HA) test 
Reagents 
1. Isotonic saline buffered with phosphate (0.05M) to pH 7.0-7.4. 
2. Red blood cells (RBC) taken and pooled from a minimum of three 
specific pathogen free chickens (if not available blood may be 
taken from birds regularly monitored and shown to be free of Avian 
Influenza antibodies) into an equal volume of Alsever's solution. 
Cells should be washed three times in PBS before use. For the test 
a 1% suspension (packed cell v/v) is PBS is recommended. 
3. The Community Reference Laboratory will supply or recommend H5 and 
H7 viruses of low virulence for use as standard antigens. 
Procedure 
1. Dispensed 0.025ml PBS into each well of a plastic microtitre plate 
(V-bottomed wells should be used). 
2. Place 0.025ml of virus suspension (ie. allantoic fluid) in the 
first we 11. 
3. Use a microtitration diluter to make two-fold dilutions (1:2 to 
1:4096) of virus across the plate. 
4. Dispense a further 0.025ml of PBS to each well. 
5. Add 0.025ml of 1% red blood cells to each well. 
-u-
6. Mix by tapping gently and place at 4°C. 
7. Plates are read 30-40 minutes later when Red Blood Cells control 
are settled. Reading Is done by tilting the plate and observing the 
presence or absence of tear-shaped streaming of the RBCs. Wells 
with no HA should flow at the same rate as the control cells with 
no virus. 
8. The HA titre is the highest dilution that causes agglutination of 
the RBCs. That dilution may be regarded as containing one HA unit 
(HAU). A more accurate method for determining the HA titre is to do 
HA tests on virus from a close range of initial dilutions ie. 1:3, 
1:4, 1:5, 1:6, etc. This is recommended for the accurate 
preparation of antigen for haemagglutination inhibition tests 
(Chapter 6). 
CHAPTER 6: Haemaoglutination Inhibition (HI) Test 
Reagents 
1. Phosphate Buffer Solution (PBS). 
2. Virus containing allantoic fluid diluted with PBS to contain 4 or 8 
HAU per 0.025ml. 
3. 1% chicken RBCs. 
4. Negative control chicken serum. 
5. Positive control serum. 
Procedure 
1. Dispense 0.025ml PBS into all wells of a plastic microtitre plate 
(with V-bottomed wells). 
2. Place 0.025ml of serum into first well of plate. 
u-
3. Use microtktration diluter to make two-fold dilutions of serum 
across plate. 
4. Add 0.025ml of diluted allantoic fluid containing 4 or 8 HAU. 
5. Mix by tapping and place plate at 4*C for a minimum of 60 minutes 
or room temperature for a minimum of 30 minutes. 
6. Add 0.025ml 1% RBCs to all wells. 
7. Mix by gentle tapping and place at 4°C. 
8. Plates are read after 30-40 minutes when control RBCs are settled. 
This is done by tilting and observing the presence or absence of 
tear-shaped streaming at the same rate as control wells containing 
RBCs (0.025ml) and PBS (0.05ml) only. 
9. The HI titre is the highest dilution of antiserum causing complete 
inhibition of 4 or 8 units of virus (an HA titration to confirm the 
presence of the required HAU should be included in each test). 
10. The validity of the results is dependent on obtaining a titre of 
less than 23 for 4 HAU or 22 for 8 HAU with the negative 
control serum and a titre of within one dilution of the known titre 
of the positive control serum. 
CHAPTER 7; Intravenous Pathogenicity Index MVP I) 
1. Infective allantoic fluid from the lowest passage level available, 
preferably from the initial isolation without any selection, is 
diluted 10~1 in sterile isotonic saline. 
2. 0.1ml diluted virus is injected intravenously into each of 10 six-
week-old chickens (specific pathogen free birds should be used). 
3. Birds are examined at 24 hour intervals for 10 days. 
-n-
4. At each observation each bird is recorded normal (0), sick (1), 
severely sick (2) or dead (3). 
5. Record results and calculate index as shown in this example: 
Day after inoculation 
Clinical Signs Total Score 
1 23456789 10 
Normal 10 200000000 12 xO-0 
Sick 0420000000 6x1-6 
Severely sick* 0 2 2 2 0 0 0 0 0 0 6x2- 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. 
-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. 
-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. 
-ÎO-
ANNEX H 
LIST OF NATIONAL AVIAN INFLUENZA LABORATORIES 
- Belgium 
- Denmark 
- Germany 
France 
- Greece 
- Ireland 
- Italy 
- Luxembourg 
- Netherlands 
- Portugal 
- Spain 
- United Kingdom Institut National de Recherches 
Vétérinaires, Groeselenberg 99, 
B 1180 Bruxelles 
National Veterinary Laboratory, 
Poultry Disease Division, HangcJvej 2 
DK 8200 Aarhus N. 
Institut fur Kleintierzucht der 
Bundesforschungsanstalt fur Land-
wi rtschaft, Braunschweig-Vôikenrode, 
Postfach 280, D 3100 Celle 
Centre National d'Etudes Vétérinaires 
et Alimentation - Laboratoire Central 
de Recherches Agricoles et Porcines, 
B.P. 53, F 22440 Ploufragan 
Veterinary Research Laboratory, 
Abbotstown, Castleknock, Dublin 15 
Istituto Patolgie Aviaire, Facolta 
di Medicina Veterinaria, Universita 
di Napoli, Via Aniezzo Falcone 394, 
I 80127 Napoli F Delpino 1 
Institut National de Recherches 
Vétérinaires, Groeselenberg 99, 
B 1180 Bruxelles 
Centraal Diergeneeskundig Instituut, 
Vestiging Virologie, Hourtibweg 39, 
NL 8221 RA Lelystad 
Laboratôrio Nactional de 
Investigaçao Veterinaria (LNIV), 
Estrada de Benfica 701, 1500 Lisboa 
Central Veterinary Laboratory, New 
Haw, Weybridge, GB-Surrey KT15 3NB 
ANNEX 111 
NAME OF THE COMMUNITY REFERENCE LABORATORY FOR AVIAN INFLUENZA 
Central Veterinary Laboratory 
New Haw 
Weybridge 
Surrey KT15 3NB 
UNITED KINGDOM 
ISSN 0254-1475 
COM(91) 304 final 
DOCUMENTS 
EN 03 
Catalogue number : CB-CO-91-344-EN-C 
ISBN 92-77-74817-6 
Office for Official Publications of the European Communities 
L-2985 Luxembourg