1994D0577 — EN — 19.01.2004 — 002.001 — 1
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►B COMMISSIONDECISION
of15July1994
concerning animal health conditions and veterinary certification for the importation of bovine
semenfromthirdcountries
(94/577/EC)
(OJ L 221, 26.8.1994, p. 26)
Amended by:
Official Journal
No page date
►M1Commission Decision 1999/495/EC of 1 July 1999 L 192 56 24.7.1999
►M2Commission Decision 2004/52/EC of 9 January 2004 L 10 67 16.1.2004
▼B
COMMISSIONDECISION
of15July1994
concerning animal health conditions and veterinary certification
fortheimportationofbovinesemenfromthirdcountries
(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 requirementsapplicable to intra-Community trade in, and imports of semen of domestic animals of thebovine species (
1), aslas t amended by Directive 93/60/EEC (2), and, in
particular, Articles10 and 11 thereof,
Whereasthe lis t of third countriesfrom which Member Statesauthorize
importation of semen of domestic animals of the bovine species (here-
inafter referred to as ‘the third countries list’) was established byCommission Decision 90/14/EEC (
3), asamended by Decis ion 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 conditionsand veterinary
certification for the importation of bovine semen from the United
Statesof America, Canada, Poland, Finland, New Zealand, Aus tria,Switzerland, Sweden, Hungary, Norway and Czechoslovakia respec-tively;
WhereasDirective 93/60/EEC which hasto be implemented in the
Member Statesbefore 1 July 1994 at the lates t amendsDirective 88/
407/EEC thereby necessitating amendment to the above third country
Decisions; whereas in order to simplify and clarify Community legisla-tion in this area it is necessary to group together the above Decisionsand to repeal those in force for each individual country;
Whereas, given the provisions of the European Economic Area Agree-
ment, it is not necessary to maintain the animal health certificate for
importation of bovine semen from Austria, Finland, Norway andSweden;
Whereasin accordance with the opinion of the Scientific Veterinary
Committee, an additional test 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 itis necessary to allow a transitional period for introduction of this Deci-1994D0577 — EN — 19.01.2004 — 002.001 — 2
(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.
▼B
sion 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;
Whereasit appearsthat the animal health s ituation in the countrieson
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 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 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 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 ofcertificates from this Decision and to ensure that all relevant certifi-cates, derogations and laboratory findings on which certification mayhave been based remain an official file for at least 12 months followingthe 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 collec-
tion centresfor the export of bovine s emen to the European EconomicCommunity asrequired by Article 9 of Directive 88/407/EEC;
Whereasanimal health conditionsand veterinary certification mus t be
adapted according to the animal health 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
conformsto the conditionss et out in the certificatesin Part 1 of
AnnexesA, B, C and D and only from thos e third countrieswhichappear in Part 2 of AnnexesA, B, C and D res pectively.
However, Member Statess hall authorize from Canada, importsof
bovine semen collected and processed in accordance with Decision91/549/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 subpara-
graph of Article 1 shall apply from 1 January 1995.
Article 4
This Decision is addressed to the Member States.1994D0577 — EN — 19.01.2004 — 002.001 — 3
▼B
ANNEX A
PART 1
ANIMALHEALTHCERTIFICATE
forimportationofbovinesemen
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▼B
1994D0577 — EN — 19.01.2004 — 002.001 — 5
▼B
►(1) (2)M11994D0577 — EN — 19.01.2004 — 002.001 — 6
▼M2
PART 2
ListofcountriesapprovedtousethemodelanimalhealthcertificateatPart
IofAnnexA
ISO code Country Remark
CH Switzerland
NZ New Zealand
RO RomaniaCY Cyprus(*)
CZ Czech Republic (*)
EE Estonia (*)
HU Hungary (*)
LT Lithuania (*)
LV Latvia (*)
MT Malta (*)
PL Poland (*)
SI Slovenia (*)
SK Slovakia (*)
(*) Only applicable until thisacceding State becomesa Member State of the Community.1994D0577 — EN — 19.01.2004 — 002.001 — 7
▼B
ANNEX B
PART 1
ANIMALHEALTHCERTIFICATE
forimportationofbovinesemenfromtheUnitedStatesofAmerica
1994D0577 — EN — 19.01.2004 — 002.001 — 8
▼B
1994D0577 — EN — 19.01.2004 — 002.001 — 9
▼B
►(1) (2)M11994D0577 — EN — 19.01.2004 — 002.001 — 10
▼B
PART 2
List of countries authorized to use the model animal health certificate at
Part1ofAnnexB
UNITED STATES OF AMERICA1994D0577 — EN — 19.01.2004 — 002.001 — 11
▼B
ANNEX C
PART 1
ANIMALHEALTHCERTIFICATE
forimportationofbovinesemen
1994D0577 — EN — 19.01.2004 — 002.001 — 12
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1994D0577 — EN — 19.01.2004 — 002.001 — 13
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►(1) (2)M11994D0577 — EN — 19.01.2004 — 002.001 — 14
▼B
PART 2
List of countries authorized to use the model animal health certificate at
Part1ofAnnexC
CANADA: excluding the Okanagan Valley region of British Columbia which is
defined asthe area enclos ed by a line drawn from a point on theCanada/United Statesborder 120º 15 ′longitude, 49º latitude north-
erly to a point 119º 35 ′longitude, 50º 30 ′latitude north easterly to a
point 119º longitude, 50º 45 ′latitude southerly to a point on the
Canada/United Statesborder 118º 15 ′longitude and 49º latitude.1994D0577 — EN — 19.01.2004 — 002.001 — 15
▼B
ANNEX D
PART 1
ANIMALHEALTHCERTIFICATE
forimportationofbovinesemen
1994D0577 — EN — 19.01.2004 — 002.001 — 16
▼B
1994D0577 — EN — 19.01.2004 — 002.001 — 17
▼B
►(1) (2)M11994D0577 — EN — 19.01.2004 — 002.001 — 18
▼B
PART 2
Listofthirdcountriesauthorizedtousethemodelanimalhealthcertificate
atPart1ofAnnexD
AUSTRALIA1994D0577 — EN — 19.01.2004 — 002.001 — 19
▼B
ANNEX E
Protocol for a competitive Elisa (‘enzyme-linked immunosorbent assay’),
test using a group specific monoclonal antibody for the detection of blue-
tonguevirusantibodies
THE BLUETONGUE COMPETITIVE ELISA USING MONOCLONAL
ANTIBODY3-17-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 asdes cribed 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 super-
natant) stored at − 20 ºC 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 ºC.
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 aliquotsand kept in the dark at − 20 ºC, 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. Orbital shaker.
9. Elisa plate reader (the test may be read visually).1994D0577 — EN — 19.01.2004 — 002.001 — 20
▼B
TEST FORMAT
1994D0577 — EN — 19.01.2004 — 002.001 — 21
▼B
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 controlRow 2 A — H is the monoclonal antibody control and consists of BTV antigen,
monoclonal antibody and conjugate. Thisrepres entsa negative control. The
mean of the optical density readings from this control row represent the 0 %
inhibition value.
Positive controlRow 3 A — H is the positive control. This consists of BTV antigen, BTV posi-
tive 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 seraIn the test format shown above, 18 sera can be tested over a dilution rate of 1/2,
1/4, 1/8 and 1/16. Thiswill give s ome 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 µl to all wells of the Elisa plate. Tap sides of plate to disperse
antigen.
2. Incubate at 37 ºC for 60 minutes on an orbital shaker. Wash plates three
timesby flooding and emtying the wellswith uns terile PBS and blot dry on
absorbent paper.
3. Add 50 µl 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 µl to all wellsof the plate except for the
blank control.
5. Incubate at 37 ºC 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
µl to all wellsof the plate.
7. Incubate at 37 ºC 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 µl of 30 % hydrogen
peroxide to each 25 ml of OPD. Add 50 µl to all wellsof the plate. Allow
colour to develop for approximately 10 minutesand s top the reaction with 1
M sulphuric acid (50 µl per well). Colour should develop in the MAB control
wellsand in thos e wellscontaining s era 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 valuesus ing the following formula:
Percentage Inhibition Value ¼100/C0
OD in the presence of test serum
OD in the absence of test serum/C2100
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.1994D0577 — EN — 19.01.2004 — 002.001 — 22
▼B
PREPARATIONOFBTVELISAANTIGEN
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'smedium.
2. Incubate at 37 ºC 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 buffer
may be added to reduce thiseffect.
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 ºC 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
ºC over a 5 ml cushion of 40 % sucrose (w/v in PBS) using 30 ml Beck-
mann centrifuge tubesand 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 ºC.
TITRATIONOFBTVELISAANTIGEN
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 isasfollows :
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 ºC on an orbital shaker.
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 ºC on an orbital shaker.6. Wash plates three times with PBS.
7. Add 50 µl of rabbit anti-mouse globulin conjugated to horseradish peroxi-
dase, diluted to a pre-titrated optimal concentration, to each well of the
microtitre plate.
8. Incubate for 1 hour at 37 ºC on an orbital shaker.9. Add substrate and chromogen as described previously. Stop the reaction after
10 minutesby the addition of 1 Molar s ulphuric acid (50 µl/well).
In the competitive assay, the monoclonal antibody must be in excess, therefore a
dilution of antigen ischos en which fallson the titration curve (not on the
plateau region) which givesapproximately 0,8 OD after 10 minutes .1994D0577 — EN — 19.01.2004 — 002.001 — 23
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Protocol for anagar-gel immunodiffusiontest forthe detection ofepizootic
haemorrhagicdiseaseantibodies
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. Thismay be achieved by any s tandard protein
concentration procedure; virusin the antigen may be inactivated by the addi-
tion 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 interna-
tional 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 radius3 mm.
3. The central well isfilled with the s tandard antigen. Peripheral wells2, 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 doesnot form a s pecific line with the antigen and it doesnot
bend the line of the control serum. Petri dishes should be examined against a
dark background and using indirect illumination.1994D0577 — EN — 19.01.2004 — 002.001 — 24