The authorization issued by the competent authority to transport eggs from a suspect holding subject to the provisions of Article 4 (2) (e) to an establishment approved for the manufacture and processing of egg products in accordance with the provisions of Article 6 (1) of Directive 89/437/EEC, hereinafter called the designated establishment, must meet the following conditions:
in order to be allowed to be removed from a suspect undertaking, eggs must:
comply with the requirements laid down in Chapter IV of the Annex to Directive 89/437/EEC;
be sent directly from the suspect undertaking to the designated establishment; each consignment must be sealed before dispatch by the official veterinarian of the suspect holding and must remain sealed throughout transport to the designated establishment;
the official veterinarian of the suspect undertaking shall inform the competent authority of the designated establishment of his intention of sending eggs to it;
the competent authority responsible for the designated establishment shall ensure that:
eggs referred to in 1 (b) will be kept isolated from other eggs from the time they arrive until they are processed;
the shells of such eggs shall be regarded as high-risk material in accordance with Article 2 (2) of Directive 90/667/EEC(1) and shall be dealt with in accordance with the requirements of Chapter II of that Directive;
the packaging material, the vehicles used to transport eggs referred to in 1 (b) and all premises with which the eggs come into contact are cleaned and disinfected in such a way as to destroy all Newcastle disease virus;
the official veterinarian of the suspect holding shall be informed of all consignments of processed eggs.
As soon as the carcases of the poultry have been removed for disposal, those parts of the premises in which the poultry was housed and any parts of other buildings, yards etc. contaminated during slaughter or post-mortem examination should be sprayed with disinfectants approved for use in accordance with Article 11 of this Directive.
Any tissue of poultry or eggs which could have contaminated buildings, yards, utensils etc. should be carefully collected and disposed of with the carcases.
The used disinfectant must remain on the surface for at least 24 hours.
Grease and dirt should be removed from all surfaces by the application of a degreasing agent and washed with water.
After washing with water as described in (a), further spraying with disinfectant should be applied.
After seven days the premises should be treated with a degreasing agent, rinsed with cold water, sprayed with disinfectant and rinsed again with water.
Used litter and manure must be treated by a method capable of killing the virus. This method must comprise at least one of the following practices:
incineration or steam treatment at a temperature of 70 °C;
burying deep enough to prevent access by vermin and wild birds;
stacking and dampening (if necessary to facilitate fermentation), covering to keep in the heat so that a temperature of 20 °C is attained and leaving covered for 42 days so as to prevent access by vermin and wild birds.
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.
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.
The organs and tissues listed in paragraph 1 may be pooled, but separate treatment of faecal material is essential. Swabs should be placed in sufficient antibiotic medium to ensure full immersion. Faeces samples and organs should be homogenized (in an enclosed blender or using a pestle and mortar and sterile sand) in antibiotic medium and made to 10-20 % w/v 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 g for 10 minutes).
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 (PBS) 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.
The clarified supernatent 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 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.
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 an haemagglutination inhibition test as described in Chapters 5 and 6. Positive inhibition 1. 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.
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 monoclonal antibody techniques, has enabled grouping of the strains and isolates. In particular, some monoclonal antibodies are available which are specific for the vaccinal strains used on the territory of the 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.
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 Community in keeping with the functions and duties of the reference laboratory.
Rapid tests for the detection of Newcastle disease virus in vaccinated birds and the detection of antibodies in unvaccinated birds are outlined below:
Several rapid test 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.
The majority of laboratories involved in Newcastle disease diagnosis are familiar with the haemagglutination 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.
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.
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 4 or 8 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 24 or greater and for 8 HAU a positive serum is any showing a titre of 23 or greater.
Dispense 0,025 ml PBS into each well of a plastic microtitre plate (V-bottomed wells should be used).
Place 0,025 ml of virus suspension (i.e. allantoic fluid) in the first well.
Use a microtitration diluter to make two-fold dilutions (1:2 to 1:4096) of virus across the plate.
Dispense a further 0,025 ml of PBS to each well.
Add 0,025 ml of 1 % red blood cells to each well.
Mix by tapping gently and place at 4 °C.
Plates are read 30 — 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.
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 (see Chapter 6).
Phosphate buffered saline (PBS)
Virus-containing allantoic fluid diluted with PBS to contain 4 or 8 HAU per 0,025 ml.
1 % chicken RBCs.
Negative control chicken serum.
Positive control serum.
Dispense 0,025 ml PBS into all wells of a plastic microtitre plate (with V-bottomed wells).
Place 0,025 ml of serum into first well of plate.
Use microtitration diluter to make two-fold dilutions of serum across plate.
Add 0,025 ml of diluted allantoic fluid containing 4 or 8 HAU.
Mix by tapping and place plate at 4 °C for a minimum of 60 minutes or room temperature for a minimum of 30 minutes.
Add 0,025 ml 1 % RBCs to all wells.
Mix by gentle tapping and place at 4 °C.
Plates are read after 30-40 minutes when control RBCs are settled. This is done by tilting and observing the presence or absence of tear-shaped streaming at the same rate as control wells containing RBCs (0,025 ml) and PBCs (0,05 ml) only.
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).
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.
| Index is a mean score per bird per observation = 112/80 = 1,4 | ||||||||||
| Clinical signs | Day after inoculation(number of birds) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | Total | Score | |
| normal | 10 | 4 | 0 | 0 | 0 | 0 | 0 | 0 | 14 × 0 | = 0 |
| sick | 0 | 6 | 10 | 4 | 0 | 0 | 0 | 0 | 20 × 1 | = 20 |
| dead | 0 | 0 | 0 | 6 | 10 | 10 | 10 | 10 | 46 × 2 | = 92 |
| Total = 112 | ||||||||||
Textual Amendments
F1 Deleted by Council Directive 2008/73/EC of 15 July 2008 simplifying procedures of listing and publishing information in the veterinary and zootechnical fields and amending Directives 64/432/EEC, 77/504/EEC, 88/407/EEC, 88/661/EEC, 89/361/EEC, 89/556/EEC, 90/426/EEC, 90/427/EEC, 90/428/EEC, 90/429/EEC, 90/539/EEC, 91/68/EEC, 91/496/EEC, 92/35/EEC, 92/65/EEC, 92/66/EEC, 92/119/EEC, 94/28/EC, 2000/75/EC, Decision 2000/258/EC and Directives 2001/89/EC, 2002/60/EC and 2005/94/EC (Text with EEA relevance).
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Council Directive 90/667/EEC of 27 November 1990 laying down the veterinary rules for the elimination and processing of animal waste, for its placing on the market and for the prevention of pathogens in feedstuffs of animal or fish origin, and amending Directive 90/425/EEC (OJ No L 363, 27.12.1990, p. 51).