1994D0577 — EN — 05.07.1999 — 001.001 — 1
This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents
►B 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)
(OJ L 221, 26.8.1994, p. 26)
Amended by:
OfficialJournal
No page date
►M1 Commission Decision 1999/495/EC of 1 July 1999 L 192 56 24.7.1999
1994D0577 — EN — 05.07.1999 — 001.001 — 2
▼B
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 CouncilDirective 88/407/EEC of 14 June 1988 l aying
down the animal health requirements applicable to intra-Communitytrade in, and imports of semen of domestic animals of the bovinespecies (
1), as last amended by Directive 93/60/EEC (2), and, in partic-
ular, 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 (herein-after referred to as ‘the third countries list')was established by Commis-sion Decision 90/14/EEC (
3), as amended by Decision 91/276/EEC (4);
Whereas the disease situation in Israell ed 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 Statesof America, Canada, Poland, Finland, New Zealand, Austria, Switzer-land, 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 countryDecisions; whereas in order to simplify and clarify Community legisla-tion in this area it is necessary to group together the above Decisionsand to repealthose in force for each individualcountry;
Whereas, given the provisions of the European Economic Area Agree-
ment, it is not necessary to maintain the animalheal th certificate forimportation of bovine semen from Austria, Finland, Norway andSweden;
Whereas in accordance with the opinion of the Scientific Veterinary
Committee, an additionaltest for epizootic haemorrhagic disease isrequired;
Whereas only in respect of Canada are routine precollection tests for
this disease carried out at intervals of six months; whereas therefore it isnecessary to allow a transitional period for introduction of this Decisionin respect of Canada to allow the Canadian veterinary authorities suffi-cient time to introduce the new test in their routine programme;
(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.
1994D0577 — EN — 05.07.1999 — 001.001 — 3
▼B
Whereas, moreover, following recent advances in the understanding ofthe epidemiology of bluetongue, importation of bovine semen fromAustralia may now be permitted;
Whereas it appears that the animalheal th situation in the countries on
the third countries list is good and controlled by well-structured andorganized veterinary services as regards diseases transmissible throughsemen;
Whereas the responsible veterinary authorities of the third countries
have undertaken to notify the Commission and the Member States bytelex or telefax, within 24 hours, of the confirmation of the occurrenceof any of the diseases contained in List A of the Office Internationald’Epizooties (OIE) or of any change in vaccination policy concerningany of them or, within an appropriate period, of the occurrence ofdiseases in List B of the OIE and any proposed change in the importrules 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 ofcertificates from this Decision and to ensure that all relevant certificates,derogations and laboratory findings on which certification may havebeen based remain an official file for at least 12 months following thedispatch 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 collec-tion centres for the export of bovine semen to the European EconomicCommunity as required by Article 9 of Directive 88/407/EEC;
Whereas animalheal th conditions and veterinary certification must be
adapted according to the animalheal th situation of the third countryconcerned;
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 ofAnnexes A, B, C and D and only from those third countries whichappear 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 Decision91/549/EEC until31 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 subpara-
graph of Article 1 shall apply from 1 January 1995.
Article 4
This Decision is addressed to the Member States.
1994D0577 — EN — 05.07.1999 — 001.001 — 4
▼B
ANNEX A
PART 1
ANIMAL HEALTH CERTIFICATE
for importation of bovine semen

1994D0577 — EN — 05.07.1999 — 001.001 — 5
▼B
13. I the undersigned officialveterinarian have read and am famil iar with CouncilDirective 88/407/EEC a s
amended and certify that:
(a) ............................................................................................................................ .....
(name of exporting country)
has been free from rinderpest and foot and mouth disease during the 12 months immediately prior t o
collection of the semen for export and until its date of dispatch and no vaccination against these disease s
has taken place during the same period;
(b) the semen collection centre at which the semen to be exported was collected was:
(i) approved under the conditions laid down in Annex A, Chapter I of Council Directive 88/407/EEC;
(ii) operated and supervised under the conditions laid down in Annex A, Chapter II of Council Directiv e
88/407/EEC;
(iii) during the period commencing 30 days prior to the date of collection of the semen to be exported unti l
30 days after collection (in the case of fresh semen until day of dispatch) have been free from rabies,
tuberculosis, brucellosis, anthrax and contagious bovine pleuropneumonia;
(c) the bovine animals standing at the semen collection centre:
(i) come from herds and/or were born to dams which satisfy the conditions at Annex B, Chapter I o f
Directive 88/407/EEC;
(ii) have, prior to entry into isolation, undergone the tests required by Annex B, Chapter I of Directiv e
88/407/EEC;
(iii) have satisfied the pre-entry isolation and testing requirements laid down in Annex B, Chapter I o f
Directive 88/407/EEC;
(iv) if resident for at least one year have undergone the routine tests according to Annex B, Chapter II o f
Directive 88/407/EEC;
(d) the semen to be exported:
(i) was obtained from donors which have been resident in
............................................................................................................................
(name of exporting country)
for the period of six months immediately prior to collection of the semen for export and which satisf y
the conditions laid down in Annex C of Directive 88/407/EEC;
(ii) was obtained from donors which are:
— standing in a semen collection centre in which all the bovine animals have within the 12 month s
prior to collection of the semen for export been subjected with negative results to a seru m
neutralization test or an Elisa for infectious bovine rhinotracheitis/infectious pustular vulvo vagi -
nitis (1),
or
— negative to a serum neutralization test or an Elisa for infectious bovine rhinotracheitis/infectiou s
pustular vulvovaginitis carried out within the 12 months prior to collection of semen for export (1),
or
— seropositive having been vaccinated in accordance with Annex B Chapter II of CouncilDirectiv e
88/407/EEC and having given a negative result to a serum neutralization test or an Elisa carrie d
out in the approved semen collection centre prior to vaccination (1);
(1) Delete as appropriate.
1994D0577 — EN — 05.07.1999 — 001.001 — 6
▼B
►(1)◄
►(2)◄
(iii) was processed, stored and transported under conditions which satisfy the terms of Directive 88 /
407/EEC.
Done at .......................................................... ,
(place)on ................................................................
(date)
/SS74(Stamp) (2)Signature (2): .........................................................................
Name in block letters: .. ............................................................
Officialtitl e: .. .......................................................................
►(2)◄
(2) The signature and the stamp must be in a colour different to that of the printing.
►(1)M1
►(2)M1
1994D0577 — EN — 05.07.1999 — 001.001 — 7
▼B
PART 2
List of countries authorized to use the mode animal health certificate at Part
1 of Annex A
CZECH REPUBLICHUNGARYNEW ZEALANDPOLANDROMANIASWITZERLANDSLOVAK REPUBLIC
1994D0577 — EN — 05.07.1999 — 001.001 — 8
▼B
ANNEX B
PART 1
ANIMAL HEALTH CERTIFICATE
for importation of bovine semen from the United States of America

1994D0577 — EN — 05.07.1999 — 001.001 — 9
▼B
13. I the undersigned officialveterinarian have read and am famil iar with CouncilDirective 88/407/EEC a s
amended and certify that:
(a) ....................................................................................... has been free from rinderpest an d
(name of exporting country)
foot and mouth disease during the 12 months immediately prior to collection of the semen for export an d
untilits date of dispatch and no vaccination against these diseases has taken pl ace during the same period;
(b) the semen collection centre at which the semen to be exported was collected was:
(i) approved under the conditions laid down in Annex A, Chapter I of Council Directive 88/407/EEC;
(ii) operated and supervised under the conditions laid down in Annex A, Chapter II of Council Directiv e
88/407/EEC;
(iii) during the period commencing 30 days prior to the date of collection of the semen to be exported unti l
30 days after collection (in the case of fresh semen until day of dispatch) have been free from rabies,
tuberculosis, brucellosis, anthrax and contagious bovine pleuropneumonia;
(c) the bovine animals standing at the semen collection centre:
(i) come from herds and/or were born to dams which satisfy the conditions at Annex B, Chapter I o f
Directive 88/407/EEC;
(ii) have, prior to entry into isolation, undergone the tests required by Annex B, Chapter I of Directiv e
88/407/EEC;
(iii) have satisfied the pre-entry isolation and testing requirements laid down in Annex B, Chapter I o f
Directive 88/407/EEC;
(iv) if resident for at least one year have undergone the routine tests according to Annex B, Chapter II o f
Directive 88/407/EEC;
(d) the semen to be exported:
(i) was obtained from donors which have been resident in
............................................................................................................................
(name of country)
for the period of six months immediately prior to collection of the semen for export and which satisf y
the conditions laid down in Annex C of Directive 88/407/EEC;
(ii) was obtained from donors which are:
— standing in a semen collection centre in which all the bovine animals have within the 12 month s
prior to collection of the semen for export been subjected with negative results to a seru m
neutralization test or an Elisa for infectious bovine rhinotracheitis/infectious pustular vulvo vagi -
nitis (1),
or
— negative to a serum neutralization test or an Elisa for infectious bovine rhinotracheitis/infectiou s
pustular vulvovaginitis carried out within the 12 months prior to collection of semen for export (1),
or
— seropositive having been vaccinated in accordance with Annex B Chapter II of CouncilDirectiv e
88/407/EEC and having given a negative result to a serum neutralization test or an Elisa carrie d
out in the approved semen collection centre prior to vaccination (1);
(1) Delete as appropriate.
1994D0577 — EN — 05.07.1999 — 001.001 — 10
▼B
►(1)◄
►(2)◄
(iii) was processed, stored and transported under conditions which satisfy the terms of Directive 88 /
407/EEC.
(iv) was obtained from donor bulls which were subjected on two occasions not more than 12 months apar t
to the following precollection and post-collection tests with negative results in an approved laborator y
(the post-collection test must be performed on a blood sample taken not less than 21 days followin g
the collection of semen for export):
— a competitive Elisa for bluetongue in accordance with Annex E,
— an agar-gelimmuno-diffusion test in accordance with Annex E and a virus neutral ization test fo r
all serotypes of epizootic haemorrhagic disease (EHD) known to exist in the exporting countr y
Done at .......................................................... ,
(place)on ................................................................
(date)
/SS74(Stamp) (2)Signature (2): .........................................................................
Name in block letters: .. ............................................................
Officialtitl e: .. .......................................................................
►(2)◄
(2) The signature and the stamp must be in a colour different to that of the printing.
►(1)M1
►(2)M1
1994D0577 — EN — 05.07.1999 — 001.001 — 11
▼B
PART 2
List of countries authorized to use the model animal health certificate at
Part 1 of Annex B
UNITED STATES OF AMERICA
1994D0577 — EN — 05.07.1999 — 001.001 — 12
▼B
ANNEX C
PART 1
ANIMAL HEALTH CERTIFICATE
for importation of bovine semen

1994D0577 — EN — 05.07.1999 — 001.001 — 13
▼B
13. I, the undersigned officialveterinarian have read and am famil iar with CouncilDirective 88/407/EEC a s
amended and certify that:
(a) ............................................................................................................................ .....
(name of exporting country)
has been free from rinderpest and foot and mouth disease during the 12 months immediately prior t o
collection of the semen for export and until its date of dispatch and no vaccination against these disease s
has taken place during the same period;
(b) the semen collection centre at which the semen to be exported was collected was:
(i) approved under the conditions laid down in Annex A, Chapter I of Council Directive 88/407/EEC;
(ii) operated and supervised under the conditions laid down in Annex A, Chapter II of Council Directiv e
88/407/EEC;
(iii) during the period commencing 30 days prior to the date of collection of the semen to be exported unti l
30 days after collection (in the case of fresh semen until day of dispatch) have been free from rabies,
tuberculosis, brucellosis, anthrax and contagious bovine pleuropneumonia;
(c) the bovine animals standing at the semen collection centre:
(i) come from herds and/or were born to dams which satisfy the conditions at Annex B, Chapter I o f
Directive 88/407/EEC;
(ii) have, prior to entry into isolation, undergone the tests required by Annex B, Chapter I of Directiv e
88/407/EEC;
(iii) have satisfied the pre-entry isolation and testing requirements laid down in Annex B, Chapter I o f
Directive 88/407/EEC;
(iv) if resident for at least one year have undergone the routine tests according to Annex B, Chapter II o f
Directive 88/407/EEC;
(d) the semen to be exported:
(i) was obtained from donors which have been resident in
............................................................................................................................
(name of exporting country)
for the period of six months immediately prior to collection of the semen for export and which satisf y
the conditions laid down in Annex C of Directive 88/407/EEC;
(ii) was obtained from donors which are:
— standing in a semen collection centre in which all the bovine animals have within the 12 month s
prior to collection of the semen for export been subjected with negative results to a seru m
neutralization test or an Elisa for infectious bovine rhinotracheitis/infectious pustular vulvo vagi -
nitis (1),
or
— negative to a serum neutralization test or an Elisa for infectious bovine rhinotracheitis/infectiou s
pustular vulvovaginitis carried out within the 12 months prior to collection of semen for export (1),
or
— seropositive having been vaccinated in accordance with Annex B Chapter II of CouncilDirectiv e
88/407/EEC and having given a negative result to a serum neutralization test or an Elisa carrie d
out in the approved semen collection centre prior to vaccination (1);
(1) Delete as appropriate.
1994D0577 — EN — 05.07.1999 — 001.001 — 14
▼B
►(1)◄
►(2)◄
(iii) was processed, stored and transported under conditions which satisfy the terms of Directive 88 /
407/EEC.
(iv) was obtained from donor bulls which were either:
— standing in the approved centre on lJul y 1994,
or
— introduced into the approved centre after 1 July 1994 having been subjected to the following test s
for bluetongue and epizootic haemorrhagic disease in an approved laboratory with negative result s
prior to entry and in either case were subjected every six months to the following tests in a n
approved laboratory with negative results:
— a competitive Elisa for bluetongue in accordance with Annex E;— an agar-gelimmuno-diffusion test in accordance with Annex E and a virus neutral ization test fo
r
all serotypes of epizootic haemorrhagic disease found in the exporting country.
Done at .......................................................... ,
(place)on ................................................................
(date)
/SS74(Stamp) (2)Signature (2): .........................................................................
Name in block letters: .. ............................................................
Officialtitl e: .. .......................................................................
►(2)◄
(2) The signature and the stamp must be in a colour different to that of the printing.
►(1)M1
►(2)M1
1994D0577 — EN — 05.07.1999 — 001.001 — 15
▼B
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 theCanada/United States border 120 °15 /prime1longitude, 49 °latitude north-
erly to a point 119 °35 /prime1longitude, 50 °30 /prime1latitude north easterly to a
point 119 °longitude, 50 °45 /prime1latitude southerly to a point on the
Canada/United States border 118 °15 /prime1longitude and 49 °latitude.
1994D0577 — EN — 05.07.1999 — 001.001 — 16
▼B
ANNEX D
PART 1
ANIMAL HEALTH CERTIFICATE
for importation of bovine semen

1994D0577 — EN — 05.07.1999 — 001.001 — 17
▼B
13. I, the undersigned officialveterinarian have read and am famil iar with CouncilDirective 88/407/EEC a s
amended and certify that:
(a) ....................................................................................... has been free from rinderpest an d
(name of exporting country)
foot and mouth disease during the 12 months immediately prior to collection of the semen for export an d
untilits date of dispatch and no vaccination against these diseases has taken pl ace during the same period;
(b) the semen collection centre at which the semen to be exported was collected was:
(i) approved under the conditions laid down in Annex A, Chapter I of Council Directive 88/407/EEC;
(ii) operated and supervised under the conditions laid down in Annex A, Chapter II of Council Directiv e
88/407/EEC;
(iii) during the period commencing 30 days prior to the date of collection of the semen to be exported unti l
30 days after collection (in the case of fresh semen until day of dispatch) have been free from rabies,
tuberculosis, brucellosis, anthrax and contagious bovine pleuropneumonia;
(c) the bovine animals standing at the semen collection centre:
(i) come from herds and/or were born to dams which satisfy the conditions at Annex B, Chapter I o f
Directive 88/407/EEC;
(ii) have, prior to entry into isolation, undergone the tests required by Annex B, Chapter I of Directiv e
88/407/EEC;
(iii) have satisfied the pre-entry isolation and testing requirements laid down in Annex B, Chapter I o f
Directive 88/407/EEC;
(iv) if resident for at least one year have undergone the routine tests according to Annex B, Chapter II o f
Directive 88/407/EEC;
(d) the semen to be exported:
(i) was obtained from donors which have been resident in
............................................................................................................................
(name of exporting country)
for the period of six months immediately prior to collection of the semen for export and which satisf y
the conditions laid down in Annex C of Directive 88/407/EEC;
(ii) was obtained from donors which are:
— standing in a semen collection centre in which all the bovine animals have within the 12 month s
prior to collection of the semen for export been subjected with negative results to a seru m
neutralization test or an Elisa for infectious bovine rhinotracheitis/infectious pustular vulvo vagi -
nitis (1),
or
— negative to a serum neutralization test or an Elisa for infectious bovine rhinotracheitis/infectiou s
pustular vulvovaginitis carried out within the 12 months prior to collection of semen for export (1),
or
— seropositive having been vaccinated in accordance with Annex B Chapter II of CouncilDirectiv e
88/407/EEC and having given a negative result to a serum neutralization test or an Elisa carrie d
out in the approved semen collection centre prior to vaccination (1);
(1) Delete as appropriate.
1994D0577 — EN — 05.07.1999 — 001.001 — 18
▼B
►(1)◄
►(2)◄
(iii) was processed, stored and transported under conditions which satisfy the terms of Directive 88 /
407/EEC.
(iv) was obtained from donor bulls which were subjected on two occasions not more than 12 months apar t
to the following pre-collection and post-collection tests with negative results in an approved labora -
tory (the post-collection test must be performed on a blood sample taken not less than 21 day s
following the collection of semen for export):
— a competitive Elisa for bluetongue in accordance with Annex E,— an agar-gelimmuno-diffusion test in accordance with Annex E and a virus neutral ization test fo
r
all serotypes of epizootic haemorrhagic disease (EHD) known to exist in the exporting country,
— a serum neutralization test for Akabane virus.
Done at .......................................................... ,
(place)on ................................................................
(date)
/SS74(Stamp) (2)Signature (2): .........................................................................
Name in block letters: .. ............................................................
Officialtitl e: .. .......................................................................
►(2)◄
(2) The signature and the stamp must be in a colour different to that of the printing.
►(1)M1
►(2)M1
1994D0577 — EN — 05.07.1999 — 001.001 — 19
▼B
PART 2
List of third countries authorized to use the model animal health certificate
at Part 1 of Annex D
AUSTRALIA
1994D0577 — EN — 05.07.1999 — 001.001 — 20
▼B
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 testserum dilutions. Antibodies to BTV present in the test serum block the reactivityof the monoclonal antibody (MAB) and result in a reduction in the expectedcolour 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 superna-
tant) 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 ofdi-sodium hydrogenorthophosphate made up to 500 ml with distilled water,divided into 25 mlal iquots and kept in the dark at − 20 °C, with 12 µl/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. Orbitalshaker.9. Elisa plate reader (the test may be read visually).
1994D0577 — EN — 05.07.1999 — 001.001 — 21
▼B
TEST FORMAT
TEST PROTOCOL
Blank control
R o w1A—Hi sab la n k controlconsisting of BTV antigen and conjugate. This
may be used to blank the Elisa reader.
MAB control
R o w2A—Hi st h e monoclonal antibody control and consists of BTV antigen,
monoclonal antibody and conjugate. This represents a negative control. Themean of the opticaldensity readings from this controlrow represent the 0 %inhibition value.
Positive control
R o w3A—Hi st h e positive control. This consists of BTV antigen, BTV
positive antiserum dilutions, MAB and conjugate. This is included as an indi-cator that the test is functioning properly and similar levels of inhibition shouldbe obtained from test to test.
1994D0577 — EN — 05.07.1999 — 001.001 — 22
▼B
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 thetest sera. The dilution range could be extended further to obtain serum dilutionend-point titres. Alternatively, for large-scale serological surveys, sera could betested at a single dilution (1/4) or two dilutions (1/2 and 1/4) as a rapid screeningtest.
PROCEDURE
1. Dilute BTV antigen to pre-titrated concentration in PBS, sonicate briefly to
disperse aggregated virus (if sonicator is not available, pipette vigorously) andadd 50 µl to all wells of the Elisa plate. Tap sides of plate to disperse antigen.
2. Incubate at 37 °C for 60 minutes on an orbitalshaker. Wash pl ates three times
by flooding and emtying the wells with unsterile PBS and blot dry onabsorbent paper.
3. Add 50 µlper wel lof bl ocking buffer. Add test sera and positive serum to the
appropriate wells and dilute across the plate using a multichannel pipette. Donot 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 µl to all wells of the plate except for the
blank control.
5. Incubate at 37 °C for 60 minutes on an orbitalshaker. Wash three times with
PBS and blot dry.
6. Dilute rabbit anti-mouse concentrate to 1/5000 in blocking buffer and add 50
µl to all wells of the plate.
7. Incubate at 37 °C for 60 minutes on an orbitalshaker. Wash three times with
PBS and blot dry.
8. Thaw the OPD and immediately before use add 12 µlof 30 % hydrogen
peroxide to each 25 mlof OPD. Add 50 µl to all wells of the plate. Allow
colour to develop for approximately 10 minutes and stop the reaction with 1M sulphuric acid (50 µl 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 aspercentage inhibition values using the following formula:
Percentage Inhibition Value = 100 − OD in the presence of test serumOD 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 easilydiscernible 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-freeEagle’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 stillattached cells from the glass.
4. Centrifuge at 2 000 — 3 000rpm 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 buffermay 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 mlof l ysis buffer.
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9. Sonicate and clarify, storing the supernatant at each stage, at total of three
times.
10. Poolthe supernatants and centrifuge at 24 000 rpm for 120 minutes at + 4
°Co v e ra5m l 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 antibody3-17-A3. The protocol is as follows:
PROCEDURE
1. Dilute BTV antigen in PBS across the microtitre plate in a two-fold dilution
series (50 µl/well) using a multichannel pipette.
2. Incubate for 1 hour at 37 °C on an orbitalshaker.
3. Wash plates three times with PBS.4. Add 50 µl 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 orbitalshaker.
6. Wash plates three times with PBS.7. Add 50 µl of rabbit anti-mouse globulin conjugated to horseradish peroxidase,
diluted to a pre-titrated optimal concentration, to each well of the microtitreplate.
8. Incubate for 1 hour at 37 °C on an orbitalshaker.
9. Add substrate and chromogen as described previously. Stop the reaction after
10 minutes by the addition of 1 Molar sulphuric acid (50 µl/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 plateauregion) 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 haemorrhagicdisease virus. BKH or vero cells are recommended. Antigen is present in thesupernatant fluid at the end of virus growth but requires 50- to 100-foldconcentration to be effective. This may be achieved by any standard proteinconcentration procedure; virus in the antigen may be inactivated by theaddition of 0,3 % (v/v) beta-propiolactone.
2. Known positive controlserum
Using the internationalreference serum and antigen a nationalstandard serum
is produced, standardized for optimalproportion against the internationalreference serum, freeze-dried and used as the known controlserum in eachtest.
3. Test serum.
PROCEDURE
1. 1 % agarose prepared in borate or sodium barbitolbuffer, 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 roundit in a circle of radius 3 mm.
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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 testsera.
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 isnegative if it does not form a specific line with the antigen and it does not bendthe line of the control serum. Petri dishes should be examined against a darkbackground and using indirect illumination.