COMMISSION DECISION of 15 July 1994 concerning animal health conditions and veterinary certification for the importation of bovine semen from third countries (94/577/EC)

THE COMMISSION OF THE EUROPEAN COMMUNITIES,

Having regard to the Treaty establishing the European Community,

Having regard to Council Directive 88/407/EEC of 14 June 1988 laying down the animal health requirements applicable to intra-Community trade in, and imports of semen of domestic animals of the bovine species (1), as last amended by Directive 93/60/EEC (2), and, in particular, Articles 10 and 11 thereof,

Whereas the list of third countries from which Member States authorize importation of semen of domestic animals of the bovine species (hereinafter referred to as 'the third countries list') was established by Commission Decision 90/14/EEC (3), as amended by Decision 91/276/EEC (4);

Whereas the disease situation in Israel led to a ban on imports from that country by Commission Decision 91/277/EEC (5);

Whereas Commission Decisions 91/479/EEC (6), 91/549/EEC (7), 92/123/EEC (8), 92/124/EEC (9), 92/125/EEC (10), 92/126/EEC (11), 92/127/EEC (12), 92/128/EEC (13), 92/386/EEC (14), 92/387/EEC (15) and 92/445/EEC (16) lay down the animal health conditions and veterinary certification for the importation of bovine semen from the United States of America, Canada, Poland, Finland, New Zealand, Austria, Switzerland, Sweden, Hungary, Norway and Czechoslovakia respectively;

Whereas Directive 93/60/EEC which has to be implemented in the Member States before 1 July 1994 at the latest amends Directive 88/407/EEC thereby necessitating amendment to the above third country Decisions; whereas in order to simplify and clarify Community legislation in this area it is necessary to group together the above Decisions and to repeal those in force for each individual country;

Whereas, given the provisions of the European Economic Area Agreement, it is not necessary to maintain the animal health certificate for importation of bovine semen from Austria, Finland, Norway and Sweden;

Whereas in accordance with the opinion of the Scientific Veterinary Committee, an additional test for epizootic haemorrhagic disease is required;

Whereas only in respect of Canada are routine precollection tests for this disease carried out at intervals of six months; whereas therefore it is necessary to allow a transitional period for introduction of this Decision in respect of Canada to allow the Canadian veterinary authorities sufficient time to introduce the new test in their routine programme;

Whereas, moreover, following recent advances in the understanding of the epidemiology of bluetongue, importation of bovine semen from Australia may now be permitted;

Whereas it appears that the animal health situation in the countries on the third countries list is good and controlled by well-structured and organized veterinary services as regards diseases transmissible through semen;

Whereas the responsible veterinary authorities of the third countries have undertaken to notify the Commission and the Member States by telex or telefax, within 24 hours, of the confirmation of the occurrence of any of the diseases contained in List A of the Office International d'Epizooties (OIE) or of any change in vaccination policy concerning any of them or, within an appropriate period, of the occurrence of diseases in List B of the OIE and any proposed change in the import rules concerning domestic animals or the semen or embryos thereof;

Whereas the responsible veterinary authorities of the countries on the third countries list have undertaken to supervise officially the issue of certificates from this Decision and to ensure that all relevant certificates, derogations and laboratory findings on which certification may have been based remain an official file for at least 12 months following the dispatch of the semen to which they refer;

Whereas the responsible veterinary authorities of the countries on the third countries list have undertaken to approve officially semen collection centres for the export of bovine semen to the European Economic Community as required by Article 9 of Directive 88/407/EEC;

Whereas animal health conditions and veterinary certification must be adapted according to the animal health situation of the third country concerned;

Whereas the measures provided for in this Decision are in accordance with the opinion of the Standing Veterinary Committee,

HAS ADOPTED THIS DECISION:

Article 1

Member States shall authorize the importation of bovine semen which conforms to the conditions set out in the certificates in Part 1 of Annexes A, B, C and D and only from those third countries which appear in Part 2 of Annexes A, B, C and D respectively.

However, Member States shall authorize from Canada, imports of bovine semen collected and processed in accordance with Decision 91/549/EEC until 31 December 1994.

Article 2

Decisions 91/479/EEC, 92/123/EEC, 92/124/EEC, 92/125/EEC, 92/126/EEC, 92/127/EEC, 92/128/EEC, 92/386/EEC, 92/387/EEC and 92/445/EEC are repealed.

Decision 91/549/EEC is repealed from 1 January 1995.

Article 3

The provisions in relation to Canada referred to in the first subparagraph of Article 1 shall apply from 1 January 1995.

Article 4

This Decision is addressed to the Member States.

Done at Brussels, 15 July 1994.

For the Commission

René STEICHEN

Member of the Commission

(1) OJ No L 194, 22. 7. 1988, p. 10.

(2) OJ No L 186, 28. 7. 1993, p. 28.

(3) OJ No L 8, 11. 1. 1990, p. 71.

(4) OJ No L 135, 30. 5. 1991, p. 58.

(5) OJ No L 135, 30. 5. 1991, p. 60.

(6) OJ No L 258, 16. 9. 1991, p. 1.

(7) OJ No L 298, 29. 10. 1991, p. 6.

(8) OJ No L 48, 22. 2. 1992, p. 1.

(9) OJ No L 48, 22. 2. 1992, p. 10.

(10) OJ No L 48, 22. 2. 1992, p. 19.

(11) OJ No L 48, 22. 2. 1992, p. 28.

(12) OJ No L 48, 22. 2. 1992, p. 37.

(13) OJ No L 48, 22. 2. 1992, p. 46.

(14) OJ No L 204, 21. 7. 1992, p. 13.

(15) OJ No L 204, 21. 7. 1992, p. 22.

(16) OJ No L 247, 27. 8. 1992, p. 1.

ANNEX A

PART 1 PART 2 List of countries authorized to use the mode animal health certificate at Part 1 of Annex A CZECH REPUBLIC

HUNGARY

NEW ZEALAND

POLAND

ROMANIA

SWITZERLAND

SLOVAK REPUBLIC

ANNEX B

PART 1 PART 2 List of countries authorized to use the model animal health certificate at Part 1 of Annex B UNITED STATES OF AMERICA

ANNEX C

PART 1 PART 2 List of countries authorized to use the model animal health certificate at Part 1 of Annex C CANADA: excluding the Okanagan Valley region of British Columbia which is defined as the area enclosed by a line drawn from a point on the Canada/United States border 120° 15& prime; longitude, 49° latitude northerly to a point 119° 35& prime; longitude, 50° 30& prime; latitude north easterly to a point 119° longitude, 50° 45& prime; latitude southerly to a point on the Canada/United States border 118° 15& prime; longitude and 49° latitude.

ANNEX D

PART 1 PART 2 List of third countries authorized to use the model animal health certificate at Part 1 of Annex D AUSTRALIA

ANNEX E

Protocol for a competitive Elisa ('enzyme-linked immunosorbent assay'), test using a group specific monoclonal antibody for the detection of bluetongue virus antibodies THE BLUETONGUE COMPETITIVE ELISA USING MONOCLONAL ANTIBODY 3-17-A3 The test is capable of detecting antibodies to all known serotypes of bluetongue virus (BTV).

The principle of the test is the interruption of the reaction between BTV antigen and a group-specific monoclonal antibody (3-17-A3) by the addition of test serum dilutions. Antibodies to BTV present in the test serum block the reactivity of the monoclonal antibody (MAB) and result in a reduction in the expected colour development on addition of enzyme substrate.

MATERIALS AND REAGENTS

1. Flat-bottomed microtitre plates.

2. Antigen: prepared as described below.

3. Blocking buffer: 5 % (w/v) 'Marvel' dried milk powder, 0,1 % (v/v) Tween-20 (supplied as polyoxyethylene sorbiton monolaurate syrup) in phosphate-buffered saline (PBS).

4. Monoclonal antibody: 3-17-A3 (supplied as hybridoma tissue-culture supernatant) stored at 20 °C or freeze-dried, diluted 1/50 with blocking buffer before use, directed against the group-specific polypeptide p7.

5. Conjugate: rabbit anti-mouse globulin (absorbed and eluted) conjugated to horseradish peroxidase and kept in the dark at 4 °C.

6. Substrate and chromogen: 0,2 gm of orthophenylene diamine (OPD) dissolved in a buffer consisting of 2,553 gm of citric acid and 4,574 gm of di-sodium hydrogenorthophosphate made up to 500 ml with distilled water, divided into 25 ml aliquots and kept in the dark at - 20 °C, with 12 ml/25 ml of hydrogen peroxide (30 % w/v) added immediately before use.

HANDLE OPD WITH CARE - WEAR RUBBER GLOVES - SUSPECTED MUTAGEN.

7. 1 Molar sulphuric acid: 26,6 ml of acid added to 473,4 ml of distilled water.

REMEMBER - ALWAYS ADD ACID TO WATER, NEVER WATER TO ACID.

8. Orbital shaker.

9. Elisa plate reader (the test may be read visually).

TEST PROTOCOL

Blank control

Row 1 A - H is a blank control consisting of BTV antigen and conjugate. This may be used to blank the Elisa reader.

MAB control

Row 2 A - H is the monoclonal antibody control and consists of BTV antigen, monoclonal antibody and conjugate. This represents a negative control. The mean of the optical density readings from this control row represent the 0 % inhibition value.

Positive control

Row 3 A - H is the positive control. This consists of BTV antigen, BTV positive antiserum dilutions, MAB and conjugate. This is included as an indicator that the test is functioning properly and similar levels of inhibition should be obtained from test to test.

Test sera

In the test format shown above, 18 sera can be tested over a dilution rate of 1/2, 1/4, 1/8 and 1/16. This will give some indication of the titre of antibody in the test sera. The dilution range could be extended further to obtain serum dilution end-point titres. Alternatively, for large-scale serological surveys, sera could be tested at a single dilution (1/4) or two dilutions (1/2 and 1/4) as a rapid screening test.

PROCEDURE

1. Dilute BTV antigen to pre-titrated concentration in PBS, sonicate briefly to disperse aggregated virus (if sonicator is not available, pipette vigorously) and add 50 ml to all wells of the Elisa plate. Tap sides of plate to disperse antigen.

2. Incubate at 37 °C for 60 minutes on an orbital shaker. Wash plates three times by flooding and emtying the wells with unsterile PBS and blot dry on absorbent paper.

3. Add 50 ml per well of blocking buffer. Add test sera and positive serum to the appropriate wells and dilute across the plate using a multichannel pipette. Do not add sera to the blank control or the MAB control.

4. Immediately after the addition of the test sera, dilute MAB in blocking buffer (to pre-titrated dilution) and add 50 ml to all wells of the plate except for the blank control.

5. Incubate at 37 °C for 60 minutes on an orbital shaker. Wash three times with PBS and blot dry.

6. Dilute rabbit anti-mouse concentrate to 1/5000 in blocking buffer and add 50 ml to all wells of the plate.

7. Incubate at 37 °C for 60 minutes on an orbital shaker. Wash three times with PBS and blot dry.

8. Thaw the OPD and immediately before use add 12 ml of 30 % hydrogen peroxide to each 25 ml of OPD. Add 50 ml to all wells of the plate. Allow colour to develop for approximately 10 minutes and stop the reaction with 1 M sulphuric acid (50 ml per well). Colour should develop in the MAB control wells and in those wells containing sera with NO antibody to BTV.

9. Examine and record the plates either visually or using a spectrophotometric reader.

ANALYSIS OF RESULTS

Calculate the mean OD reading from the MAB controls. This represents the 0 % inhibition value. Optical density readings from the test sera are expressed as percentage inhibition values using the following formula:

Percentage Inhibition Value = 100 OD in the presence of test serum

OD in the absence of test serum × 100

Inhibition values greater than 40 % at a serum dilution of 1/4 are considered positive. Visual reading is possible as 40 % inhibition is the lowest value easily discernible by eye.

PREPARATION OF BTV ELISA ANTIGEN 1. Wash 10 roux of confluent BHK-21 cells three times with serum-free Eagle's medium and infect with bluetongue virus serotype 1 in serum-free Eagle's medium.

2. Incubate at 37 °C and examine daily for cytopathic effect (cpe).

3. When cpe is evident in 80 - 90 % of the cell sheet of each roux, harvest the virus by shaking any still-attached cells from the glass.

4. Centrifuge at 2 000 - 3 000 rpm to pellet the cells.

5. Discard the supernatant and resuspend the cells in approximately 30 ml of PHS containing 1 % 'Sarkosyl' and 2 ml phenolmethylsulphonyl fluoride (lysis buffer). This may cause the cells to form a gel and more lysis buffer may be added to reduce this effect.

NOTE: phenylmethylsulphonyl fluoride is harmful - handle with extreme caution.

6. Disrupt the cells for 60 seconds using an ultrasonic probe at an amplitude of 30 microns.

7. Centrifuge at 10 000 rpm for 10 minutes.

8. Store the supernatant at + 4 °C and resuspend the remaining cell pellet in 10 - 20 ml of lysis buffer.

9. Sonicate and clarify, storing the supernatant at each stage, at total of three times.

10. Pool the supernatants and centrifuge at 24 000 rpm for 120 minutes at + 4 °C over a 5 ml cushion of 40 % sucrose (w/v in PBS) using 30 ml Beckmann centrifuge tubes and an SW 28 rotor.

11. Discard the supernatant, drain the tubes thoroughly and resuspend the pellet in PBS by sonication. Store the antigen in aliquots at 70 °C.

TITRATION OF BTV ELISA ANTIGEN Bluetongue Elisa antigen is titrated by the indirect Elisa. Two-fold dilutions of antigen are titrated against a constant dilution (1/50) of monoclonal antibody 3-17-A3. The protocol is as follows:

PROCEDURE

1. Dilute BTV antigen in PBS across the microtitre plate in a two-fold dilution series (50 ml/well) using a multichannel pipette.

2. Incubate for 1 hour at 37 °C on an orbital shaker.

3. Wash plates three times with PBS.

4. Add 50 ml of monoclonal antibody 3-17-A3 (diluted 1/50) to each well of the microtitre plate.

5. Incubate for 1 hour at 37 °C on an orbital shaker.

6. Wash plates three times with PBS.

7. Add 50 ml of rabbit anti-mouse globulin conjugated to horseradish peroxidase, diluted to a pre-titrated optimal concentration, to each well of the microtitre plate.

8. Incubate for 1 hour at 37 °C on an orbital shaker.

9. Add substrate and chromogen as described previously. Stop the reaction after 10 minutes by the addition of 1 Molar sulphuric acid (50 ml/well).

In the competitive assay, the monoclonal antibody must be in excess, therefore a dilution of antigen is chosen which falls on the titration curve (not on the plateau region) which gives approximately 0,8 OD after 10 minutes.

Protocol for an agar-gel immunodiffusion test for the detection of epizootic haemorrhagic disease antibodies The agar-gel immo-diffusion test is carried out according to the following protocol:

MATERIALS AND REAGENTS

1. Antigen

Precipitating antigen is prepared in any cell culture system that supports the rapid multiplication of the appropriate serotype(s) of epizootic haemorrhagic disease virus. BKH or vero cells are recommended. Antigen is present in the supernatant fluid at the end of virus growth but requires 50- to 100-fold concentration to be effective. This may be achieved by any standard protein concentration procedure; virus in the antigen may be inactivated by the addition of 0,3 % (v/v) beta-propiolactone.

2. Known positive control serum

Using the international reference serum and antigen a national standard serum is produced, standardized for optimal proportion against the international reference serum, freeze-dried and used as the known control serum in each test.

3. Test serum.

PROCEDURE

1. 1 % agarose prepared in borate or sodium barbitol buffer, pH 8,5 to 9,0 is poured into a petri dish to a minimum depth of 3,0 mm.

2. A test pattern of seven moisture-free wells, each 5,0 mm in diameter, is cut in the agar. The pattern consists of one centre well and six wells arranged round it in a circle of radius 3 mm.

1

6 2

X

5 3

4

3. The central well is filled with the standard antigen. Peripheral wells 2, 4 and 6 are filled with known positive serum, wells 1, 3 and 5 are filled with test sera.

4. The system is incubated for up to 72 hours at room temperature in a closed humid chamber.

INTERPRETATION

A test serum is positive if it forms a specific precipitation line with the antigen and forms a complete line of identity with the control serum. A test serum is negative if it does not form a specific line with the antigen and it does not bend the line of the control serum. Petri dishes should be examined against a dark background and using indirect illumination.