Council Directive of 26 June 1964 on animal health problems affecting intra-Community trade in bovine animals and swine (64/432/EEC)

[F1 CHAPTER II U.K. TESTS FOR ENZOOTIC BOVINE LEUKOSIS

Tests for enzootic bovine leukosis shall be carried out by the immune-diffusion test under the conditions described in A and B or by the enzyme-linked immunosorbent assay (Elisa) under the conditions described in C. The immune-diffusion method may only be used for individual tests. If test results are the subject of a duly-substantiated challenge, an additional check shall be carried out by means of the immune-diffusion test.

A. Agar gel immune-diffusion test for enzootic bovine leukosis U.K.

1. The antigen to be used in the test must contain bovine leukosis virus glycoproteins. The antigen must be standardized against a standard serum (El serum) supplied by the State Veterinary Serum Laboratory, Copenhagen. U.K.

2. The official institutes indicated below must be made responsible for calibrating the standard working antigen of the laboratory against the official EEC standard serum (El serum) provided by the State Veterinary Serum Laboratory, Copenhagen. U.K.

(a)

Germany: Bundesforschungsanstalt für Viruskrankheiten der Tiere, Tubingen;

(b)

Belgium: Institut national de recherches vétérinaires, Brussels;

(c)

France: Laboratoire national de pathologie bovine, Lyon;

(d)

Grand Duchy of Luxembourg:

(e)

Italy: Istituto zooprofilattico spenmentale, Perugia;

(f)

Netherlands: Instituut voor Dierhouderij en Diergezondheid (ID-DLO), Lelystad;

(g)

Denmark: Statens Veterinaere Serum Laboratorium, Copenhagen;

(h)

Ireland: Veterinary Research Laboratory, Abbotstown, Dublin;

(i)

United Kingdom:

(1)

Great Britain: Veterinary Laboratory Agency, Addlestone, Weybridge, England;

(2)

Northern Ireland: Veterinary Research Laboratory, Stormont, Belfast;

(j)

Spain: Subdirreccion general de sanidad animal. Laboratorio de sanidad y produccion animal Algete, Madrid;

(k)

Portugal: Laboratorio Nacional de Investigaçáo Veterinária, Lisbon;

(l)

Greece: Κέντρο Κτηνιατρικών Ιδρυμάτων Νεαπόλεως 25, 153 10 Αθήνα ;;

(m)

Austria: Bundesanstalt für Tierseuchenbekämpfung, Mödling;

(n)

Finland: Eläinlääkintä-ja elintarvikelaitos, Helsinki/Anstalten för veterinärmedicin och livsmedel, Helsingfors;

(o)

Sweden: Statens veterinärmedicinska anstalt, Uppsala [F2 ;]

(p)

[F3 Czech Republic: Státní veterinární ústav Praha,

Sídlištní 24/136, 165 03 Praha 6;

(q)

Estonia: Veterinaar- ja Toidulaboratoorium,

Kreutzwaldi 30, 51006 Tartu;

(r)

Cyprus: Εργαστήριο Αναφοράς για τις Ασθένειες των Ζώων, Κτηνιατρικές Υπηρεσίες, 1417 Λευκωσία National Reference Laboratory for Animal Health Veterinary Services,CY-1417 Nicosia ;

(s)

Latvia: Valsts veterinārmedicīnas diagnostikas centrs

Lejupes iela 3

LV-1076 Rīga;

(t)

Lithuania: Nacionalinė veterinarijos laboratorija,

J. Kairiūkščio g. 10,

LT-2021 Vilnius;

(u)

Hungary: Országos Állategészségügyi Intézet (OÁI),Pf. 2., Tábornok u. 2., H-1581 Budapest ;

(v)

Malta: Institute of the supplying country;

(w)

Poland: Laboratorium Zakładu Biochemii

Państwowego Instytutu Weterynaryjnego

Al. Partyzantów 57, PL-24-100 Puławy;

(x)

Slovenia: Nacionalni veterinarski inštitut, Gerbičeva 60,

SI-1000 Ljubljana;

(y)

Slovakia: Štátny veterinárny ústav, Pod dráhami 918,

SK-960 86 Zvolen.]

Textual Amendments

F2 Substituted by Act concerning the conditions of accession of the Czech Republic, the Republic of Estonia, the Republic of Cyprus, the Republic of Latvia, the Republic of Lithuania, the Republic of Hungary, the Republic of Malta, the Republic of Poland, the Republic of Slovenia and the Slovak Republic and the adjustments to the Treaties on which the European Union is founded.

F3 Inserted by Act concerning the conditions of accession of the Czech Republic, the Republic of Estonia, the Republic of Cyprus, the Republic of Latvia, the Republic of Lithuania, the Republic of Hungary, the Republic of Malta, the Republic of Poland, the Republic of Slovenia and the Slovak Republic and the adjustments to the Treaties on which the European Union is founded.

3. The standard antigens used in the laboratory must be submitted at least once a year to the EEC reference laboratories listed in 2 for testing against the official EEC standard serum. Apart from this standardization, the antigen in use can be calibrated in accordance with B. U.K.

4. The reagents for the test shall consist of: U.K.

(a)

antigen: the antigen must contain specific glycoproteins of enzootic bovine leukosis virus which has been standardized against the official EEC serum;

(b)

the test serum;

(c)

known positive control serum;

(d)

agar gel:

• 0,8 % agar,

• 8,5 % NaCl,

• 0,05 M Tris-buffer pH 7,2;

15 ml of this agar must be introduced into a petri dish of 85 mm diameter, resulting in a depth of 2,6 mm of agar.

5. A test pattern of seven moisture-free wells must be cut in the agar to the bottom of the plate; the pattern must consist of one central well and six wells in a circle around it. U.K.

Diameter of central well: 4 mm

Diameter of peripheral wells: 6 mm

Distance between central and peripheral wells: 3 mm

6. The central well must be filled with the standard antigen. Peripheral wells 1 and 4 (see diagram below) are filled with the known positive serum, wells 2, 3, 5 and 6 with the test sera. The wells must be filled until the meniscus disappears. U.K.

7. This results in the following quantities being obtained: U.K.

• antigen: 32 μl

• control serum: 73 μl

• test serum: 73 μl.

8. Incubation must be for 72 hours at room temperature (20 to 27 ^o C) in a closed humid chamber. U.K.

9. The test may be read at 24 and 48 hours but a final result may not be obtained before 72 hours: U.K.

(a)

a test serum is positive if it forms a specific precipitin line with the BLV antigen and forms a complete line of identity with the control serum;

(b)

a test serum is negative if it does not form a specific precipitin line with the BLV antigen and if it does not bend the line of the control serum;

(c)

the reaction cannot be considered conclusive if it:

(i)

bends the line of the control serum towards the BLV antigen well without forming a visible precipitin line with the antigen;

or

(ii)

if it cannot be read either as negative or as positive.

In inconclusive reactions the test may be repeated and concentrated serum utilized.

10. Any other well configuration or pattern may be utilized provided that the E4 serum diluted 1:10 in negative serum can be detected as positive. U.K.

B. Method for antigen standardization U.K.

Solutions and materials required U.K.

1. 40 ml of 1,6 % agarose in 0,05 M Tris/HCl buffer, pH 7,2 with 8,5 % NaCl; U.K.

2. 15 ml of a bovine leukosis serum, having antibody only to bovine leukosis virus glycoproteins, diluted 1:10 in 0,05 M Tris/HCl buffer, pH 7,2 with 8,5 % NaCl; U.K.

3. 15 ml of a bovine leukosis serum, having antibody only to bovine leukosis virus glycoproteins, diluted 1:5 in 0,05 M Tris/HCl buffer, pH 7,2 with 8,5 % NaCl; U.K.

4. four plastic petri dishes with a diameter of 85 mm; U.K.

5. a punch with a diameter of 4 to 6 mm; U.K.

6. a reference antigen; U.K.

7. the antigen which is to be standardized; U.K.

8. a water bath (56 ^o C). U.K.

Procedure U.K.

Dissolve the agarose (1,6 %) in the Tris/HCl buffer by carefully heating to 100 ^o C. Place in 56 ^o C water bath for approximately one hour. Also, place the bovine leukosis serum dilutions in a 56 ^o C water bath.

Now mix 15 ml of the 56 ^o C agarose solution with the 15 ml bovine leukosis serum (1:10), quickly shake and pour 15 ml into each of two petri dishes. Repeat this procedure with the bovine leukosis serum diluted 1:5.

When the agarose has hardened, holes are made in it as follows:

Addition of antigen U.K.

(i)

Petri dishes 1 and 3:

• well A — undiluted reference antigen,

• well B — 1:2 diluted reference antigen,

• wells C and E — reference antigen,

• well D — undiluted test antigen.

(ii)

Petri dishes 2 and 4:

• well A — undiluted test antigen,

• well B — 1:2 diluted test antigen,

• well C — 1:4 diluted test antigen,

• well D — 1:8 diluted test antigen.

Additional instructions U.K.

1. The experiment shall be carried out with two serum dilutions (1:5 and 1:10) in order to achieve optimal precipitation U.K.

2. If the precipitation diameter is too small with both dilutions, then the serum must be further diluted. U.K.

3. If the precipitation diameter in both dilutions is too large and faint, then a lower serum must be chosen. U.K.

4. The final concentration of the agarose must be 0,8 %; that of the sera 5 and 10 % respectively. U.K.

5. Plot the measured diameters in the following coordinate system. The dilution of the antigen to be tested with the same diameter as the reference antigen is the working dilution. U.K.

C. Enzyme-linked immunosorbent assay (Elisa) for detecting enzootic bovine leukosis U.K.

1. The material and reagents to be used are as follows: U.K.

(a)

solid-phase microplates, cuvettes or any other solid phase;

(b)

the antigen is fixed to the solid phase with or without the aid of polyclonal or monoclonal catching antibodies. If antigen is coated directly to the solid phase, all test samples giving positive reactions have to be retested against control antigen in the case of EBL. The control antigen should be identical to the antigen except for the BLV antigens. If catching antibodies are coated to the solid phase, the antibodies must not react to antigens other than BLV antigens;

(c)

the biological fluid to be tested;

(d)

a corresponding positive and negative control;

(e)

conjugate;

(f)

a substrate adapted to the enzyme used;

(g)

a stopping solution, if necessary;

(h)

solutions for the dilution of the test samples for preparations of the reagents and for washing;

(i)

a reading system appropriate to the substrate used.

2. Standardization and sensitivity of test U.K.

The sensitivity of the Elisa assay must be of such a level that E4 serum is scored positive when diluted 10 times (serum samples) or 250 times (milk samples) more than the dilution obtained of individual samples when these are included in pools. In assays where samples (serum and milk) are tested individually E4 serum diluted 1 to 10 (in negative serum) or 1 to 250 (in negative milk) must be scored positive when tested in the same assay dilution as used for the individual test samples. The official institutes listed in A.2 will be responsible for checking the quality of the Elisa method, and in particular for determining, for each production batch, the number of samples to be pooled on the basis of the count obtained for the E4 serum.

The E4 serum will be supplied by the National Veterinary Laboratory, Copenhagen.

3. Conditions for use of the Elisa test for EBL U.K.

The Elisa method may be used on a sample of milk or whey taken from the milk collected from a farm with at least 30 % of dairy cows in milk.

If this method is used, measures must be taken to ensure that the samples taken can be identified with the animals from which the milk or sera examined were taken.]

Textual Amendments

F1 Substituted by Council Directive 97/12/EC of 17 March 1997 amending and updating Directive 64/432/EEC on health problems affecting intra-Community trade in bovine animals and swine.