No C 146/12 Official Journal of the European Communities 5. 6. 91 
II 
(Preparatory Acts) 
COMMISSION 
Proposal for a Council Regulation (EEC) introducing Community measures for the control of 
Newcastle disease 
(91/C 146/14) 
COM(91) 137 final 
(Submitted by the Commission on 16 May 1991) 
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 popu­
lation; 
"Whereas, it is necessary to establish at Community level 
the control measures to be taken in the event of outbreak 
of Newcastle disease in order to ensure development of 
the poultry sector and contribute to the protection of 
animal health in the Community; 
Whereas an outbreak of Newcastle disease can quickly 
take on epizootic proportions, causing mortality and 
disturbances on a scale liable to reduce sharply the prof­
itability 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 be implemented when its presence 
is confirmed; whereas such measures must be modulated by the competent authorities to take account of whether 
or not a country carries out a prophylactic vaccination 
programme in all or part of its territory; 
Whereas it is necessary to prevent any spread of the 
disease as soon as an outbreak occurs, by carefully moni­
toring 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 the responsible laboratory, the 
coordination of which must be ensured by a Community 
reference laboratory; 
Whereas it is necessary to make provision for the 
Member States which vaccinate to draw up vaccination 
plans and to inform the Commission and other Member 
States about those plans; 
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 
Newcastle disease in poultry other than racing pigeons, 
without prejudice to the Community provisions 
governing intra-Community trade. 
2. This Regulation shall not apply in the event of 
detection of Newcastle disease in other birds. However, 
in this case, Member States shall inform the Commission 
of any measure they take. 
5.6.91 Official Journal of the European Communities No C 146/13 
Article 2 
1. For the purposes of this Regulation, the definitions 
given in Article 2 of Council Directive 90/539/EEC (') 
shall apply as appropriate. 
The following definitions shall also apply: 
(a) infected poultry means any poultry: 
— in which the presence of Newcastle disease has 
been officially confirmed following an exam­
ination by an approved laboratory, or 
— in the case of second and subsequent outbreaks 
in which clinical symptoms or post-mortem 
lesions consistent with Newcastle disease are 
present; 
(b) poultry suspected of being infected means any poultry 
showing clinical symptoms or post-mortem lesions 
which are such that the presence of Newcastle 
disease may reasonably be suspected; 
(c) poultry suspected of being contaminated means any 
poultry which may have been directly or indirectly 
exposed to the Newcastle disease virus; 
(d) swill means waste from kitchens, restaurants or, as 
the case may be, from industries using meat; 
(e) competent authority means the veterinary authority 
appointed for the purpose by the national adminis­
tration of the country concerned, being directly 
responsible to the national administration within the 
scope of this Regulation, and reporting through the 
national administration; 
(f) official veterinarian means the veterinarian 
designated by the competent authority of the 
Member State. 
Article 3 
Any suspected case of Newcastle disease shall be 
immediately notified to the competent authority. 
Article 4 
1. When poultry are suspected of being infected or 
contaminated with Newcastle disease, the official veter­
inarian 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. 
O OJ No L 303, 31. 10. 1990, p. 6. 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 number 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; 
it may be checked at each visit; 
(b) all poultry on the holding 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 holding, 
— of poultry meat or carcases, or of animal feed, 
implements, waste and litter or any other thing 
liable to transmit Newcastle disease from the 
holding, 
be prohibited unles authorized by the official veter­
inarian; 
(e) no eggs 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 points (b) to (e). 
4. The competent authority may extend 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 Newcastle 
disease has been ruled out by the official veterinarian. 
NoC 146/14 Official Journal of the European Communities 5.6.91 
Article 5 
1. As soon as the presence of Newcastle disease 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 taken: 
(a) all poultry on the holding shall 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 manner 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; this treatment, carried out in 
accordance with the instructions of the official 
veterinarian, shall ensure the destruction of any 
Newcastle disease 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) eggs laid during the presumed incubation period 
which have been moved from the holding shall, 
wherever possible, be traced and destroyed, but 
poultry which have already hatched from these eggs 
shall be placed under official surveillance; 
(e) after carrying out operations listed in (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 (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 give 
reason to suspect possible contamination. 
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 structure and size of these units and the operations 
carried out there are such that they are completely 
separate as regards housing, keeping and feeding, and 
taking into account their possible vaccinal immunity, so 
that the virus cannot spread from one to another. 2. The Commission shall, in accordance with the 
procedure laid down in Article 24, 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 Newcastle disease 
may have existed on the holding, 
— the possible origin of the Newcastle disease 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 carcases and any implement 
or substance likely to have carried Newcastle disease 
virus to or from the holding in question. 
2. In order to provide full coordination of all 
measures necessary to ensure eradication of Newcastle 
disease as quickly as possible and for the purpose of 
carrying out the epidemiological enquiry, a crisis unit 
shall be established. 
The general rules concerning national crisis units and the 
Community crisis unit fixed by Council Regulation 
.../.. ./EEC shall apply. 
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 official control shall be to detect 
immediately any suspicion of Newcastle disease, count 
the poultry and monitor their movements and, where 
appropriate, to take the action provided for in para­
graph 3. 
3. "When a holding is subject to 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 the presence of 
Newcastle disease on the holding. The above movement 
restrictions shall cover a period of a least 21 days starting 
from the latest date of potential contamination; if during 
the 21-day period it is discovered that potential contami­
nation has taken place, then the minimum period of 
restrictions shall be at least seven days. 
5. 6.91 Official Journal of the European Communities No C 146/15 
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. 
Article 9 
1. Once the diagnosis of Newcastle disease 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 kilometres and a surveillance zone based on a 
minimum radius of 10 kilometres. 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 holdings; 
(e) the control of movements of persons handling 
poultry, poultry carcases and eggs and vehicles 
carrying poultry, carcases and eggs to and from the 
holdings; 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: 
(i) of poultry for immediate slaughter to a slaugh­
terhouse 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 Council Regu­
lation (EEC) No .../... (on animal health 
conditions governing intra-Community trade in 
and imports from third country of fresh poul-
trymeat and fresh meat of reared game birds) 
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 (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 disin­
fected. 
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 disin­
fected before and after use; 
(g) a prohibition in 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. 
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 movements 
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 infected area 
designated by the competent authority. The special 
health mark provided for in Article 6 of Regulation 
(EEC) No .../... (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 infected area, unless to 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; 
No C 146/16 Official Journal of the European Communities 5.6.91 
(f) a prohibition of fairs, markets, shows or other 
gatherings of poultry and other birds; 
(g) without prejudice to the provisions of (b) and (c) the 
prohibition of transport of poultry, except for transit 
by major highways or railways. 
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. 
Article 10 
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 in a position to supply the 
competent authority with information concerning the 
movements of poultry and eggs which they have trans­
ported or marketed and to furnish all the details 
concerning such information. 
Article 11 
1. The disinfectants to be used and their concen­
trations 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 Newcastle disease virus shall be carried out 
in accordance with Annex I. 
Article 13 
1. Each Member State shall designate: 
(a) one or more national laboratories at which facilities 
and expert personnel shall be maintained to permit 
full antigenic and biological typing of Newcastle 
disease virus at all times and to confirm results 
obtained in regional diagnostic laboratories; 
(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 vaccines allowed for prophylactic use in the 
country or stock-piled for emergency use may be 
tested for efficacy, potency and purity. 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 Newcastle disease laboratories 
referred to in paragraph 2 shall be responsible for coor­
dinating the standards and diagnostic methods laid down 
in each Newcastle disease 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 Newcastle disease 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 Newcastle 
disease is named 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 ('), in accordance with the procedure laid 
down in Article 24. 
Article 15 
1. Vaccination against Newcastle disease with 
vaccines authorized by the competent authority may be 
used for a prophylactic purpose or to supplement the 
control measures carried out when the disease appears. 
2. Only vaccines which meet the following conditions 
shall be authorized: 
(a) the vaccine must conform with the provisions of the 
European Pharmacopeia and have been produced, 
tested and distributed under official control; 
(') OJ No L 224, 18. 8. 1990, p. 19. 
5. 6. 91 Official Journal of the European Communities No C 146/17 
(b) any vaccine imported from a third country shall 
satisfy the requirements laid down in (a). 
3. Further criteria for using vaccines against 
Newcastle disease may be established in accordance with 
the procedure laid down in Article 24. 
Article 16 
1. A Member State in which voluntary or compulsory 
prophylactic vaccination against Newcastle disease is 
carried out shall inform the Commission and the other 
Member States. 
2. The information given in accordance with 
paragraph 1 must specify: 
— the characteristics and composition of each vaccine 
used, 
— the procedures for supervision of the distribution, 
storage and use of vaccines, 
— the species and categories of poultry which may or 
shall be subject to vaccination, 
— the areas in which vaccination may or shall be carried 
out. 
Article 17 
1. When Newcastle disease has been confirmed, the 
competent authority, in order to supplement the other 
control measures provided for in this Regulation, may 
specify a territorial area and period in which the prompt 
and systematic vaccination (emergency vaccination) of 
designated species of poultry shall be carried out under 
official control. A Member State applying emergency 
vaccination shall inform the Commission and other 
Member States within the framework of the Standing 
Veterinary Committee, set up by Council Decision 
68/361/EEC O, about the Newcastle disease situation 
and emergency vaccination programme. 
2. In the case referred to in paragraph 1, the vacci­
nation or re-vaccination of poultry on the holding 
subject to the restrictions referred to in Article 4 is 
prohibited. 
3. In the case referred to in paragraph 1: 
(a) the designated species of poultry shall be vaccinated 
as soon as possible; 
(b) all poultry of designated species born on or trans­
ferred to a holding inside the vaccination area must 
be or have been vaccinated; 
0) OJ No L 255, 18. 10. 1968, p. 23. (c) throughout the vaccination operation provided for in 
paragraph 1 all poultry of designated species kept on 
holdings inside the vaccination area must remain 
there except: 
— day-old chicks moved to a holding inside the 
vaccination area where they shall be vaccinated, 
— poultry moved direct to a slaughterhouse for 
immediate slaughter. If the slaughterhouse is 
located outside the vaccination area the 
movement of poultry shall be permitted only after 
the official veterinarian has carried out a health 
inspection of the holding; 
(d) when the vaccination operations provided for in (a) 
have been completed movements out of the vacci­
nation area may be authorized for: 
— day-old chicks intended for meat production may 
be moved to a holding where they shall be 
vaccinated; the holding must be kept under 
surveillance until the poultry which have been 
moved have been slaughtered, 
— poultry vaccinated more than 21 days previously 
and intended for immediate slaughter, 
— hatching eggs which originate from breeding 
poultry vaccinated at least 21 days previously; the 
eggs and their packing must be disinfected before 
movement. 
4. The measures provided for in (b) and (d) of 
paragraph 3 shall be applied for a period of 3 months 
following completion of vaccination operations provided 
for in paragraph 1; they may be extended for one or 
more additional periods of three months. 
5. By way of derogation from (a) and (b) of 
paragraph 3, the competent authorities may exempt 
certain flocks of particular scientific value from 
systematic vaccination, provided that all necessary steps 
are taken by the competent authority to ensure 
protection of their health and to subject them to periodic 
serological checks. 
6. The Commission shall follow the development of 
the disease situation and if necessary it may, in 
accordance with the procedure laid down in Article 24, 
take a decision in particular in relation to control of 
movements and vaccination. 
Article 18 
1. When, in a given region, an epizootic of Newcastle 
disease is exceptionally serious and is tending to spread, 
the Member State concerned: 
No C 146/18 Official Journal of the European Communities 5. 6. 91 
— 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, 
— 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 the disease situation and 
applied control measures. 
2. The boundaries of the high health risk area may be 
revised with a progessive elimination of surveillance 
zones. The measures laid down in paragraph 1 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 State concerned, in 
particular determination of the high health risk area and 
recourse to the provisions of Article 17, must be decided 
in accordance with the procedure laid down in Article 
24. 
Article 19 
1. The use of swill originating from means of interna­
tional transport, such as ships, land vehicles, or aircraft, 
is prohibited for the feeding of poultry; such swill shall 
be collected and destroyed under official supervision. 
2. The use of swill other than as defined in paragraph 
1, or poultry scraps may be authorized for the feeding of 
poultry only after a heat-treatment ensuring the 
destruction of Newcastle disease virus. 
3. If necessary, the Commission shall, in accordance 
with the procedure laid down in Article 24, lay down the 
criteria for the implementation of paragraph 2. 
Article 20 
1. Each Member State shall draw up a contingency 
plan, specifying the national measures to be implemented 
in the event of an outbreak of Newcastle disease. 
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. 2. The criteria to be applied for drawing up the 
contingency plan shall be those laid down in Commission 
Decision 91/42/EEC (*), which apply mutatis mutandis. 
The Commission may, in accordance with the procedure 
laid down in Article 24, amend or supplement those 
criteria taking into account the specific nature of 
Newcastle disease. 
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 Regu­
lation 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 24. 
The plans may subsequently be amended or 
supplemented, in accordance with the same procedure, 
to take into account developments in the situation. 
Article 21 
Veterinary experts from the Commission may, in colla­
boration with the authorities of the Member State 
concerned, in so far 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. 
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 24. 
Article 22 
Detailed rules for financial participation by the 
Community in steps taken pursuant to this Regulation 
are laid down in Decision 90/424/EEC. 
Article 23 
The Annexes to this Regulation may be amended by the 
Commission, in accordance with the procedure laid 
down in Article 24, in particular in order to take into 
account the development in diagnostic procedures. 
O OJ No L 33, 29. 1. 1991, p. 29. 
5. 6. 91 Official Journal of the European Communities No C 146/19 
Article 24 
1. The Commission shall be assisted by the Standing 
Veterinary Committee, hereinafter referred to as 'the 
Committee'. 
2. Where the procedure laid down in this Article is to 
be followed, the following provisions shall apply. 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. 
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. 
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. Article 25 
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. 
ANNEX I 
DIAGNOSTIC PROCEDURES FOR THE CONFIRMATION AND DIFFERENTIAL DIAGNOSIS 
OF NEWCASTLE DISEASE 
The following procedures for the isolation and characterization of Newcastle disease viruses should be 
regarded as guidelines and the minima to be applied in the diagnosis of the disease. 
The virus responsible for Newcastle disease is the prototype virus of the paramyxoviridae. At present, there 
are nine serologically distinguishable groups of avian paramyxoviruses, which have been designated PMV-1 
to PMV-9. All Newcastle disease viruses are placed in the PMV-1 group. For the purpose of the diagnostic 
procedures for the confirmation and differential diagnosis of Newcastle disease the following definition 
shall apply: 
Newcastle disease means an infection of poultry caused by any avian strain of the paramyxovirus 1 with an 
intracerebral pathogenicity index (ICPI) in day-old chicks greater than 0,7. 
CHAPTER 1 
Sampling and treatment of samples 
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 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 homogenized (in an enclosed blender or using a pestle and mortar and sterile sand) in 
antibiotic medium and made to 10 to 20 °/o 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 (e.g. 800 to 1 000 x g for 10 minutes). 
No C 146/20 Official Journal of the European Communities 5. 6. 91 
3. Antibiotic medium 
Different laboratories have used various formulations of antibiotic medium with success and laboratories 
referred to in Annex II will be able to offer advice for a particular country. High concentrations of 
antibiotics are required for faeces samples 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 to 7,4. 
CHAPTER 2 
Virus isolation 
Virus isolation in embryonated fowls' eggs 
The clarified supernatent fluid should be inoculated in 0,1 to 0,2 ml amounts into the allantoic cavity of 
each of a minimum of four embryonated, fowls' eggs which have been incubated for 8 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 Newcastle disease virus. 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 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. 
CHAPTER 3 
Differential diagnosis 
1. Preliminary differentiation 
It is intended that all haemagglutinating viruses should be submitted to the national laboratory referred 
to in Annex II for full identification, characterization and pathogenicity tests. However, it is important 
that interim control measures for Newcastle disease aimed at limiting the spread of the virus should be 
implemented as soon as possible and regional laboratories should be able to identify the presence of 
Newcastle disease virus. The haemagglutinating fluids should, therefore, be used in a haemagglutination 
inhibition test as described in Chapters 5 and 6. Positive inhibition, i.e. 24, or more, with the Newcastle 
disease virus specific polyclonal antiserum of titre known to be at least 29 would serve as preliminary 
identification enabling the imposition of interim control measures. 
2. Confirmatory identification 
The national laboratory should undertake full differential diagnosis of any haemagglutinating agent. 
Confirmation of Newcastle disease virus would again be by inhibition in haemagglutination inhibition 
tests with monospecific chicken antisera. Intracerebral pathogenicity index tests as described in Chapter 
7 should be carried out on all positive isolates. Pathogenicity indices of greater than 0,7 indicate the 
presence of virus requiring the full implementation of control measures. 
Recent developments in typing Newcastle disease viruses, particularly monocional antibody techniques, 
has enabled grouping of the strains and isolates. In particular, some monocional antibodies are available 
which are specific for the vaccinal strains used on the territory of the European Community and can be 
employed in simple haemagglutination inhibition tests. Since live vaccine strains may often be isolated 
from sampled poultry the advantage of their rapid identification at the national laboratory referred to in 
Annex II is obvious. Such monoclonal antibodies should be obtained by the Community reference 
laboratory as referred to in Article 14 and supplied to the national laboratories to enable confirmation 
of the isolation of vaccinal viruses. 
The national laboratories should submit all haemagglutinating agents to the Community reference 
laboratory. 
5. 6. 91 Official Journal of the European Communities No C 146/21 
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. 
CHAPTER 4 
Rapid tests for detection of Newcastle disease virus and antibodies 
Rapid tests for the detection of Newcastle disease virus in vaccinated birds and the detection of antibodies in 
unvaccinated birds are outlined below 
1. Detection of Newcastle disease virus 
Several rapid tests that directly detect Newcastle disease antigens have been employed in the diagnosis 
of infections in vaccinated birds. Those most commonly used to date are fluorescent antibody tests on 
longitudinal sections of the trachea and peroxidase antibody tests on the brain. There seems no reason 
to doubt that other direct antigen detection tests could be applied to Newcastle disease virus infections. 
The drawback to such tests is that it is impracticable to examine all the potential sites of replication of 
Newcastle disease virus in the vaccinated birds. So that, for example, absence of evidence of virus in the 
trachea does not preclude virus replication in the gut. No direct detection method is recommended for 
routine use in the diagnosis of Newcastle disease, although in specific circumstances such tests may have 
a useful role. 
2. Detection of antibodies in unvaccinated birds 
The majority of laboratories involved in Newcastle disease diagnosis are familiar with the haemaggluti-
nation inhibition test and the recommendation described below relate to this test for the measurement of 
antibodies to the virus. However, enzyme-linked immunosorbent assays (Elisa) may be successfully used 
to detect antibodies to the virus. It is suggested that if there is a wish to employ an Elisa test at regional 
laboratory level the test should be monitored by the national laboratory referred to in Annex II. 
(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 Newcastle disease virus 
haemagglutinating antigen in standard haemagglutination inhibition tests as defined in Chapter 6. 
There is some debate as to whether four or eight haemagglutinin units should be used for the HI 
test. 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 2* or greater, for 8 HAU a positive serum is any showing 
a titre of 23 or greater. 
No C 146/22 Official Journal of the European Communities 5. 6. 91 
CHAPTER 5 
Haemagglutination (HA) Test 
1. Reagents 
(a) Isotonic saline buffered with phosphate (0,05 M) to pH 7,0 to 7,4. 
(b) 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 NDV 
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) in PBS is recommended. 
(c) NDV strain Ulster 2C is recommended for use as standard antigen. 
2. Procedure 
(a) Dispense 0,025 ml PBS into each well of a plastic microtitre plate (V-bottomed wells should be 
used). 
(b) Place 0,025 ml of virus suspension (i.e. allantoic fluid) in the first well. 
(c) Use a microtitration diluter to make two-fold dilutions (1:2 to 1:4096) of virus across the plate. 
(d) Dispense a further 0,025 ml of PBS to each well. 
(e) Add 0,25 ml of 1 % red blood cells to each well. 
(f) Mix by tapping gently and place at 4 ° C. 
(g) Plates are read 30 to 40 minutes later when controls 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. 
(h) 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 
Haemagglutination inhibition (HI) test 
1. Reagents (see Chapter 5) 
(a) PBS. 
(b) Virus-containing allantoic fluid diluted with PBS to contain 4 or 8 HAU per 0,025 ml. 
(c) 1 % chicken RBCs. 
(d) Negative control chicken serum. 
(e) Positive control serum. 
5.6.91 Official Journal of the European Communities No C 146/23 
2. Procedure 
(a) Dispense 0,025 ml PBS into all wells of a plastic microtitre plate (with V-bottomed wells). 
(b) Place 0,025 ml of serum into first well of plate. 
(c) Use microtitration diluter to make two-fold dilutions of serum accross plate. 
(d) Add 0,025 ml of diluted allantoic fluid containing 4 or 8 HAU. 
(e) Mix by tapping and place plate at 4 °C for a minimum of 60 minutes or room temperature for a 
minimum of 30 minutes. 
(0 Add 0,025 ml 1 % RBCs to all wells. 
(g) Mix by gentle tapping and place at 4 °C. 
(h) Plates are read after 30 to 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. 
(i) The HI titre is the highest dilution of antiserum causing complete inhibition of four or eight units of 
virus (a HA titration to confirm the presence of the required HAU should be included in each test). 
(j) The validity of the results is dependent on obtaining a titre of less than 25 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 
Intracerebral pathogenicity index test 
1. Infective freshly harvested allantoic fluid (HA titre must be greater than 24) is diluted 1:10 in sterile 
isotonic saline (anti-biotics must not be used). 
2. 0,05 ml of the diluted virus is injected intracerebrally into each of 10 one-day-old chicks (i.e. > 24 
hours < 40 hours after hatching). The chicks should be hatched from eggs obtained from a specific 
pathogen free flock. 
3. The birds are examined at intervals of 24 hours for eight days. 
4. At each observation each bird is scored: 0 = normal; 1 = sick; 2 = dead. 
5. The index is calculated as shown in the following example: 
Clinical signs 
Normal 
Sick 
Dead Day after inoculation 
number of birds 
1 
10 
0 
0 2 
4 
6 
0 3 
0 
10 
0 4 
0 
4 
6 5 
0 
0 
10 6 
0 
0 
10 7 
0 
0 
10 8 
0 
0 
10 Total 
14 @ 0 
20 @ 1 
46 @ 2 
TOTAL Score 
- 0 
- 20 
- 92 
= 112 
Index is mean score per bird per observation = 112/80 = 1,4. 
No C 146/24 Official Journal of the European Communities 5. 6. 91 
CHAPTER 8 
Assessment of plaque-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 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 adsorb 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 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. 
ANNEX II 
LIST OF NATIONAL NEWCASTLE DISEASE LABORATORIES 
BELGIUM Institut national de recherches veterinaires, Groeselenberg 99, 
B-1180 Bruxelles 
DENMARK National Veterinary Laboratory, Poultry Disease Division, 
Hangavej 2, DK-8200 Arhus N 
FEDERAL REPUBLIC OF GERMANY Institut fur Kleintierzucht der Bundesforschungsanstalt fiir 
Landwirtschaft, Braunschweig-Vdlkenrode, Postfach 2 80, 
D-W-3100 Celle 
FRANCE Centre national d'etudes veterinaires et alimentation — 
Laboratoire central de recherches agricoles et porcine, boite 
postale 53, F-22440 Ploufragan 
GREECE Institut de maladies infectieuses et parasitaires d'Athenes, 25 rue 
Neapoleos, 153 10, Agia Paraskevi, Attiki 
IRELAND Veterinary Research Laboratory, Abbotstown, Castleknock, 
IRL-Dublin 15 
ITALY Istituto zooprofilattico sperimentale di Padova, via G. Orus 
n. 2, 1-35100 Padova 
LUXEMBOURG Institut national de recherches veterinaires, Groeselenberg 99, 
B-1180 Bruxelles 
NETHERLANDS Centraal Diergeneeskundig Instituut, Vestiging Virologie, 
Hourtibweg 39, NL-8221 RA Lelystad 
5. 6. 91 Official Journal of the European Communities No C 146/25 
PORTUGAL 
SPAIN 
UNITED KINGDOM Laborat6rio Nacional de Investigate) Veterinaria (LNIV), 
Estrada de Benfica, 701, P-1500 Lisboa 
Laboratorio de Sanidad y Producci6n Animal, Zona Franca, 
Circcunvalaci6n — Tramo 6, Esquina Calle 3, E-08004 
Barcelona 
Central Veterinary Laboratory, New Haw, Weybridge, 
UK-Surrey KT15 3NB 
ANNEX III 
NAME OF THE COMMUNITY REFERENCE LABORATORY FOR NEWCASTLE DISEASE 
Central Veterinary Laboratory 
New Haw 
Weybridge 
UK-Surrey KT15 3NB