1964L0432 — EN — 01.05.2004 — 009.001 — 1
This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents
►B COUNCILDIRECTIVE
of26June1964
onanimalhealthproblemsaffecting intra-Communitytradeinbovineanimalsandswine
(64/432/EEC)
(OJ P 121, 29.7.1964, p. 1977)
Amended by:
Official Journal
No page date
►M1Council Directive 66/600/EEC of 25 October 1966 P 192 3294 27.10.1966
►M2Council Directive 70/360/EEC of 13 July 1970 L 157 40 18.7.1970
►M3Council Directive 71/285/EEC of 19 July 1971 L 179 1 9.8.1971
►M4Council Directive 72/97/EEC of 7 February 1972 L 38 95 12.2.1972
►M5Council Directive 72/445/EEC of 28 December 1972 L 298 49 31.12.1972
►M6Council Directive 72/462/EEC of 12 December 1972 L 302 28 31.12.1972
►M7Council Directive 73/150/EEC of 5 June 1973 L 172 18 28.6.1973
►M8Council Directive 74/387/EEC of 15 July 1974 L 202 36 24.7.1974
►M9Council Directive 75/379/EEC of 24 June 1975 L 172 17 3.7.1975
►M10Council Directive 77/98/EEC of 21 December 1976 L 26 81 31.1.1977
►M11Council Directive 79/109/EEC of 24 January 1979 L 29 20 3.2.1979
►M12Council Directive 79/111/EEC of 24 January 1979 L 29 26 3.2.1979
►M13Council Directive 80/219/EEC of 22 January 1980 L 47 25 21.2.1980
►M14Council Directive 80/1098/EEC of 11 November 1980 L 325 11 1.12.1980
►M15Council Directive 80/1102/EEC of 11 November 1980 L 325 18 1.12.1980
►M16 amended by Council Directive 85/571/EEC of 19 December 1985 L 372 12 31.12.1985
►M17Council Directive 80/1274/EEC of 22 December 1980 L 375 75 31.12.1980
►M18Council Directive 81/476/EEC of 24 June 1981 L 186 20 8.7.1981
►M19Council Directive 82/61/EEC of 26 January 1982 L 29 13 6.2.1982
►M20Council Directive 82/893/EEC of 21 December 1982 L 378 57 31.12.1982
►M21Council Directive 83/646/EEC of 13 December 1983 L 360 44 23.12.1983
►M22Council Directive 84/336/EEC of 19 June 1984 L 177 22 4.7.1984
►M23Council Directive 84/643/EEC of 11 December 1984 L 339 27 27.12.1984
►M24Council Directive 84/644/EEC of 11 December 1984 L 339 30 27.12.1984
►M25Council Directive 85/320/EEC of 12 June 1985 L 168 36 28.6.1985
►M26Council Regulation (EEC) No 3768/85 of 20 December 1985 L 362 8 31.12.1985
►M27Council Directive 85/586/EEC of 20 December 1985 L 372 44 31.12.1985
►M28Council Decision 87/231/EEC of 7 April 1987 L 99 18 11.4.1987
►M29Council Directive 87/489/EEC of 22 September 1987 L 280 28 3.10.1987
1964L0432 — EN — 01.05.2004 — 009.001 — 2
►M30Council Directive 88/406/EEC of 14 June 1988 L 194 1 22.7.1988
►M31Council Directive 89/360/EEC of 30 May 1989 L 153 29 6.6.1989
►M32Council Directive 89/662/EEC of 11 December 1989 L 395 13 30.12.1989
►M33Council Directive 90/422/EEC of 26 June 1990 L 224 9 18.8.1990
►M34Council Directive 90/423/EEC of 26 June 1990 L 224 13 18.8.1990
►M35Council Directive 90/425/EEC of 26 June 1990 L 224 29 18.8.1990
►M36Council Directive 91/499/EEC of 26 June 1991 L 268 107 24.9.1991
►M37Council Directive 91/687/EEC of 11 December 1991 L 377 16 31.12.1991
►M38Council Directive 92/65/EEC of 13 July 1992 L 268 54 14.9.1992
►M39Council Directive 92/102/EEC of 27 November 1992 L 355 32 5.12.1992
►M40Council Directive 94/42/EC of 27 July 1994 L 201 26 4.8.1994
►M41Council Directive 95/25/EC of 22 June 1995 L 243 16 11.10.1995
►M42Council Directive 97/12/EC of 17 March 1997 L 109 1 25.4.1997
►M43 amended by Council Directive 98/99/EC of 14 December 1998 L 358 107 31.12.1998
►M44Council Directive 98/46/EC of 24 June 1998 L 198 22 15.7.1998
►M45Directive 2000/15/EC of the European Parliament and the Council of 10
April 2000L 105 34 3.5.2000
►M46Directive 2000/20/EC of the European Parliament and of the Council of
16 May 2000L 163 35 4.7.2000
►M47Commission Decision 2001/298/EC of 30 March 2001 L 102 63 12.4.2001
►M48Commission Regulation (EC) No 535/2002 of 21 March 2002 L 80 22 23.3.2002
►M49Commission Regulation (EC) No 1226/2002 of 8 July 2002 L 179 13 9.7.2002
Amended by:
►A1Act of Accession of Denmark, Ireland and the United Kingdom of Great
Britain and Northern IrelandL 73 14 27.3.1972
(adapted by Council Decision of 1 January 1973) L 2 1 1.1.1973
►A2Act of Accession of Greece L 291 17 19.11.1979
►A3Act of Accession of Austria, Sweden and Finland C 241 21 29.8.1994
(adapted by Council Decision 95/1/EC, Euratom, ECSC) L 1 1 1.1.1995
►A4Act 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 EuropeanUnion isfoundedL 236 33 23.9.2003
Corrected by:
►C1Corrigendum, OJ L 120, 13.5.1975, p. 13 (64/432/EEC)
►C2Consolidated text of corrigenda to instruments published in Special
Editions1952-72, p. 10 (64/432/EEC)
►C3Consolidated text of corrigenda to instruments published in Special
Editions1952-72, p. 20 (66/600/EEC)
►C4Corrigendum, OJ L 120, 13.5.1975, p. 15 (71/285/EEC)
►C5Consolidated text of corrigenda to instruments published in Special
Editions1952-72, p. 90 (71/285/EEC)
►C6Corrigendum, OJ L 64, 10.3.1977, p. 28 (77/98/EEC)
►C7Corrigendum, OJ L 49, 24.2.1981, p. 16 (80/1098/EEC)
1964L0432 — EN — 01.05.2004 — 009.001 — 3
►C8Corrigendum, OJ L 329, 17.11.1981, p. 22 (80/1102/EEC)
►C9Corrigendum, OJ L 192, 2.7.1982, p. 23 (82/61/EEC)
►C10Corrigendum, OJ L 133, 22.5.1985, p. 32 (84/643/EEC)
►C11Corrigendum, OJ L 42, 13.2.1985, p. 20 (84/644/EEC)
▼B
COUNCILDIRECTIVE
of26June1964
on animal health problems affecting intra-Community trade in
bovineanimalsandswine
(64/432/EEC)
THE COUNCIL OF THE EUROPEAN ECONOMIC COMMUNITY,
Having regard to the Treaty establishing the European Economic
Community, and in particular Articles43 and 100 thereof;
Having regard to the proposal from the Commission;
Having regard to the Opinion of the European Parliament (1);
Having regard to the Opinion of the Economic and Social
Committee (2);
Whereas Council Regulation No 20 on the progressive establishment of
a common organisation of the market in pigmeat (3) isalready in force
and a similar regulation is to be adopted for beef and veal and whereasthese regulations also concern trade in live animals;
Whereas Regulation No 20 substitutes for the numerous traditional
means of protection at the frontier a single system designed in parti-cular to facilitate intra-Community trade; whereasthe regulation to beadopted for beef and veal is also designed to eliminate obstacles tosuch trade;
Whereas, so long as intra-Community trade in bovine animals and
swine is hindered by differences between the health requirements ofMember States, the implementation of the above-mentioned regulationswill not have the desired effect;
Whereas, to eliminate those differences, measures must be taken within
the framework of the common agricultural policy and in line with regu-lations already adopted or in preparation on the progressive
establishment of a common organisation of markets; whereas the
animal health provisions of Member States must therefore be approxi-mated;
Whereasthe right of Member Statesunder Article 36 of the Treaty to
continue to apply prohibitions or restrictions on imports, exports or
goods in transit justified on grounds of the protection of health andlife of humansand animalsnevertheles sdoesnot exempt them fromthe obligation to approximate the provisions on which those prohibi-tions and restrictions are based, in so far as the differences between
those provisions hinder the implementation and functioning of the
common agricultural policy;
Whereas, in the context of such approximation, the exporting country
must be required to ensure that bovine animals and swine for breeding,
production or slaughter intended for intra-Community trade, the places
from which those animals come and are shipped and the means oftransport used satisfy certain animal health requirements so as to ensurethat the animals are not a source of contagious or infectious disease;
Whereas, so that Member States may be sure that these requirements
are satisfied, provision must be made for the issue by an official veter-inarian of a health certificate which will accompany the animalstotheir destination;
WhereasMember Statesmus t have the right to prohibit the introduc-
tion into their territory of bovine animalsand s wine if they are foundto be suffering or are suspected of suffering from a contagious or infec-tious disease, if they may spread such disease without actually1964L0432 — EN — 01.05.2004 — 009.001 — 4
(1) OJ No 61, 19.4.1963, p. 1254/63.
(2) OJ No 121, 29.7.1964, p. 2009/64.
(3) OJ No 30, 20.4.1962, p. 945/62.
▼B
suffering from it or if they do not comply with Community animal
health provisions;
Whereasthere isno reas on to allow Member Statesto prohibit the
introduction of bovine animals and swine into their territory for reasons
other than those of animal health and whereas, therefore, the consignorshould at his own request or upon request of his representative beallowed to return the animalsto the country of export unles sthere arereasons to the contrary;
Whereas, in case of prohibition or restriction, the reasons therefor
should be made known to the consignor of the animals or his represen-
tative and to the competent central authority of the country of export sothat they be aware of the reasons why such measures were imposed;
Whereas in the event of dispute between himself and the authority of
the Member State of destination as to the justification for prohibitionor restriction, the consignor should be enabled to obtain the opinion
of a veterinary expert whom he may select from a panel drawn up by
the Commission;
Whereas in some cases and for certain categories of animals it appears
that the general provisions of this Directive may be relaxed withoutinvolving any health risk, by allowing consignee Member States togrant general or special derogations;
Whereas, in certain fields presenting special problems, the provisions
in Member Statescannot be approximated until a more thorough s tudyhasbeen made;
Whereasa s implified amendement procedure may be provided for
Annexes B to D since the rules contained in those Annexes are of atechnical nature and liable to change; whereas the Commission should
therefore be entrusted with making such amendments after consulting
the Member States;
HAS ADOPTED THIS DIRECTIVE:
Article 1
ThisDirective s hall apply to intra-Community trade in bovine animals
and swine with the exception of feral pigs as defined in Article 2 (e) of
Directive 80/217/EEC (1) without prejudice to the provisions laid down
in Directives80/215/EEC (2), 85/511/EEC, 88/407/EEC (3), 89/608/
EEC (4), 90/425/EEC, 90/429/EEC (5), 90/667/EEC (6), 91/496/EEC,
91/628/EEC (7), 92/102/EEC (8), 92/119/EEC and Decision 90/424/
EEC (9).
Article 2
1. The definitionsgiven in Article 2 of Directive 90/425/EEC and
Article 2 of Directive 91/628/EEC shall apply.1964L0432 — EN — 01.05.2004 — 009.001 — 5
(1) OJ No L 47, 21. 2. 1980, p. 11. Directive aslas t amended by Directive 93/
384/EEC (OJ No L 166, 8. 7. 1993, p. 34).
(2) OJ No L 47, 21. 2. 1980, p. 4. Directive aslas t amended by Directive 91/
687/EEC (OJ No L 377, 31. 12. 1991, p. 16).
(3) OJ No L 194, 22. 7. 1988, p. 10. Directive aslas t amended by Directive 93/
60/EEC (OJ No L 186, 28. 7. 1993, p. 28).
(4) OJ No L 351, 2. 12. 1989, p. 34.
(5) OJ No L 224, 18. 8. 1990, p. 62. Directive aslas t amended by the 1994 Act
of Accession.
(6) OJ No L 363, 27. 12. 1990, p. 51. Directive aslas t amended by Directive
92/118/EEC.
(7) OJ No L 340, 11. 12. 1991, p. 17. Directive aslas t amended by Directive
95/29/EC (OJ No L 148, 30. 6. 1995, p. 52).
(8) OJ No L 355, 5. 12. 1992, p. 32. Directive aslas t amended by the 1994 Act
of Accession.
(9) OJ No L 224, 18. 8. 1990, p. 19. Decision as last amended by Decision 94/
370/EC (OJ No L 168, 2. 7. 1994, p. 31).▼M42
▼M42
2. In addition the following definitionsapply for the purpos esof this
Directive:
(a)herdmeansan animal or group of animalskept on a holding
(within the meaning of Article 2 (b) of Directive 92/102/EEC) as
an epidemiological unit; if more than one herd iskept on aholding, each of these herds shall form a distinct unit and shallhave the same health status;
(b)animal for slaughter meansa bovine animal (including the s pecies
Bison bison andBubalus bubalus ) or swine intended to be taken to
a slaughterhouse or assembly centre from which it may only moveto slaughter;
(c)animals for breeding or production meansbovine animals
(including the species Bison bison andBubalus bubalus ) and swine
other than those referred to in (b), including those intended forbreeding, milk or meat production, or draft purposes, shows orexhibition with the exception of animalstaking part in cultural
and sporting events;
(d)officially tuberculosis-free bovine herd meansa bovine herd which
satisfies the conditions laid down in ►
M43Annex A.I, para-
graphs1 and 2 ◄;
(e)officially tuberculosis-free Member State orregion of a Member
Statemeansa Member State or part of a Member State which
satisfies the conditions laid down in ►M43Annex A.I, para-
graphs4 and 5 ◄;
(f)officially brucellosis-free bovine herd meansa bovine herd which
satisfies the conditions laid down in ►M43Annex A.II, para-
graphs1 and 2 ◄;
(g)officially brucellosis-free region meansa region of a Member
State which satisfies the conditions laid down in Annex A.II, para-graphs7, 8 and 9;
(h)officially brucellosis-free Member State meansa Member State
which satisfies the conditions laid down in ►
M43Annex A.II,
paragraphs7, 8 and 9 ◄;
(i)brucellosis-free bovine herd means a bovine herd which satisfies
the conditionslaid down in ►M43Annex A.II, paragraphs4 and
5◄;
(j)officially enzootic-bovine-leukosis-free herd meansa herd which
satisfies the conditions laid down in Annex D, Chapter I, Sections
A and B;
(k)officially enzootic-bovine-leukosis-free Member State orregion
meansa region or Member State which meetsthe requirements
laid down in ►M43Annex D, Chapter I, SectionsE and F ◄;
(l)official veterinarian meansthe veterinarian appointed by the
competent authority of the Member State;
(m)approved veterinarian meansany veterinarian approved by the
competent authority in accordance with the provisions of Article14 (3) (B);
(n)compulsorily notifiable diseases means the diseases listed in
Annex E (I);
(o)assembly centre means holdings, collection centres and markets, at
which bovine animalsor s wine originating from different holdings
are grouped together to form consignments of animals intended fortrade. These assembly centres must be approved for tradingpurposes and meet the requirements laid down in Article 11;
(p)region meansthat part of a Member State'sterritory which isat
least 2 000 km
2in area and which is subject to inspection by the
competent authoritiesand includesat leas t one of the followingadministrative regions:
— Belgium: province — provincie
— Germany: Regierungsbezirk
— Denmark: amt or island1964L0432 — EN — 01.05.2004 — 009.001 — 6
▼M42
— France: département
— Italy: provincia
— Luxemburg —
— Netherlands: rrv-kring
— United Kingdom: England, Walesand Northern Ireland: county
Scotland: district or island area
— Ireland: county
— Greece: νοµός
— Spain: provincia
— Portugal: continente: distrito, and other parts of Portugal's
territory: região autónoma
— Austria: Bezirk
— Sweden: län
— Finland: lääni/län
— Czech Republic: kraj
— Estonia: maakond
— Cyprus: επαρχία (district)
— Latvia: rajons
— Lithuania: apskritis
— Hungary: megye
— Malta: —
— Poland: powiat
— Slovenia: obmo čje
— Slovakia: kraj;
(q)dealer means any natural or legal person who buys and sells
animalscommercially either directly or indirectly, who hasaregular turnover of these animals and who within a maximum of
30 days of purchasing animals resells them or relocates them
from the first premises to other premises not within his ownershipand meetsthe conditionslaid down in Article 13.
Article 3
1. Each Member State shall ensure that only animals that fulfil the
relevant conditionslaid down in thisDirective are s ent from itsterri-
tory to that of another Member State.
2. Bovine animals and swine covered by this Directive must:
(a) be subjected:
— to an identity check, and
— to a clinical inspection within 24 hours of departure by an offi-
cial veterinarian and show no clinical sign of disease;
(b) not have been obtained from a holding or an area which for health
reasons is subject to prohibition or restriction affecting the speciesinvolved in accordance with Community and/or national legisla-
tion;
(c) be identified asprovided for in Directive 92/102/EEC;
(d) not be animals which are to be slaughtered and not be restricted
under a contagious or infectious disease eradication programme ofa Member State or region;
(e) comply with the provisions of Articles 4 and 5.1964L0432 — EN — 01.05.2004 — 009.001 — 7
▼A4
▼M42
▼M42
Article 4
1. Bovine animals and swine covered by this Directive must at no
time between leaving the holding of origin and arriving at destinationcome into contact with cloven-hoofed animalsother than animalsthathave the same health status.
2. Bovine animals and swine covered by this Directive must be
transported in means of transport meeting the requirements of Directive91/628/EEC and in addition the requirementsof Article 12.
3. Rules for the approval of sites where cleansing and disinfection
may be carried out shall be determined in accordance with the proce-
dure set out in Article 17.
Article 5
1. Bovine animals and swine covered by this Directive must be
accompanied during transportation to destination by a health►
M43certificate conforming to either model 1 or 2 set out in Annex
F asappropriate ◄. The certificate shall consist of a single sheet or,
where more than one page is required, shall be in such a form that
any two or more pagesare part of an integrated whole and indivis ibleand shall contain a serial number. It shall be drawn up on the day ofthe health inspection, in one of the official languages of the countryof destination at least. The certificate shall be valid for 10 days fromthe date of the health examination.
2. The health inspection for the issuing of the health certificate
(including additional guarantees) for a consignment of animals may be
carried out in the holding of origin or of an assembly centre. For this
purpose the competent authority shall ensure that any health certificateis drawn up by the official veterinarian after inspections, visits andcontrolsasprovided by thisDirective.
However asregards :
(a) animals coming from approved assembly centres, such certification
shall be:
— on the basis of an official document containing the necessary
information completed by the official veterinarian for theholding of origin, or
— in the form of the ►
M43certificate according to either model
1 or 2 in Annex F asappropriate ◄with SectionsA and B
duly completed and certified by the official veterinarian for theholding of origin;
(b) animalscoming from an approved holding which isparticipating in
the surveillance network provided for in Article 14, such certifica-
tion shall be:
— on the basis of an official document containing the necessary
information completed by the approved veterinarian for theholding of origin, or
— in the form of the ►
M43certificate according to either model
1 or 2 in Annex F asappropriate ◄with SectionsA and B
duly completed and certified by the approved veterinarian forthe holding of origin.
For this purpose, the official veterinarian will ensure where appropriate
that the additional guaranteesprovided for in Community legis lationare fulfilled.
3. The official veterinarian for the assembly centre shall carry out
all necessary checks on animals arriving there.
4. The official veterinarian completing ►
M43Section C of the
certificate according to either model 1 or 2 in Annex F asappro-priate ◄, shall ensure that the movement is registered on the Animo
system on the day the certificate is issued.
5. The animalscovered by thisDirective may trans it through an
assembly centre which is located in one other Member State before1964L0432 — EN — 01.05.2004 — 009.001 — 8
▼M42
being consigned to the Member State of destination. In this case, the
►M43certificate according to either model 1 or 2 in Annex F as
appropriate ◄►M43(including Section C) ◄must be completed
by the official veterinarian responsible in the Member State where the
animals originate. The official veterinarian responsible for the assemblycentre of transit shall provide certification to the Member State ofdestination by completing a second ►
M43certificate according to
either model 1 or 2 in Annex F asappropriate ◄, endorsing it with
the serial number of the original and attaching it to the original certifi-
cate or to an officially endorsed copy thereof. In this case the combinedvalidity of the certificatess hall not exceed that provided for in para-graph 1.
Article 6
1. Animalsfor breeding or production mus t, in addition to the
requirementsof Articles3, 4 and 5:
— have remained in a single holding of origin for a period of 30 days
prior to loading, or since birth in the holding of origin where theanimals are less than 30 days old. The official veterinarian must,on the basis of the official identification provided for in Article 3
(2) (c) and official records, be satisfied that the animals have
complied with thiscondition and furthermore that the animalshave originated in the Community or have been imported from athird country in compliance with Community animal health legisla-tion.
However, in the case of animals transiting through an approved
assembly centre in the Member State of origin, the period duringwhich the assembly of these animals takes place outside the holding
of origin shall not exceed six days,
— with regard to animalsimported from a third country into a
Member State which is not that of ultimate destination, be trans-ported to the Member State of destination as quickly as practicableunder cover of a certificate issued under Article 7 of Directive 91/496/EEC,
— with regard to animalsimported from a third country upon arrival at
destination and before any further movements satisfy the require-mentsof thisDirective, and in particular the res idency requirement
in the first indent, and may not be brought into the herd until the
veterinarian responsible for that holding has ascertained that theanimals in question are not likely to jeopardize the health status ofthe holding.
If an animal from a third country isintroduced into a holding no
animal from the holding may be traded for 30 daysfollowing intro-duction unless the imported animal is isolated from all other
animalson the holding.
2. Bovine animalsfor breeding and production mus t, in addition to
the requirementsin Articles3, 4 and 5:
(a) come frome an officially tuberculosis-free bovine herd, and in the
case of animals more than six weeks old, have reacted negativelyto an intradermal tuberculin test carried out during the 30 days
prior to leaving the herd of origin, in accordance with the provi-
sions of Annex B point 32 (d).
Thisintradermal tuberculin tes t isnot required if the animalsorigi-
nate in a Member State or part of a Member State recognized as
officially tuberculosis free or in a Member State or part of a
Member State with an approved surveillance network;
(b) in the case of uncastrated animals which come from an officially
brucellosis-free bovine herd and more than 12 months old, have
shown a brucella count lower that 30 international units (IU) of
agglutination per millilitre when given a serum agglutination test(or any test approved by Standing Veterinary Committee (SVC)procedure following the adoption of the relevant protocols) carriedout during the 30 daysprior the leaving the herd of origin andcomplying with the provisions of Annex C Section A.1964L0432 — EN — 01.05.2004 — 009.001 — 9
▼M42
This serum agglutination test (or any test approved by SVC proce-
dure following the adoption of the relevant protocols) is notrequired if the animalsoriginate in a Member State or part of a
Member State recognized asofficially brucellos isfree or in a
Member State or part of a Member State with an approved surveil-lance network;
(c) come from an officially enzootic-bovine-leukosis-free herd and, if
more than 12 monthsold, have reacted negatively to an individual
test carried out during the 30 days prior to leaving the herd of
origin and complying with the provisions of Annex D.
The test is not required if the animals originate in a Member State
or part of a Member State recognized asofficially enzootic-bovine-leukosis free or in a Member State or part of a Member State with
an approved surveillance network;
(d) at no time between leaving the holding of origin and arriving at
destination come into contact with bovine animals which meetonly the requirementsin paragraph 3;
(e) until 31 December 2000, not be subject to the test requirements
laid down in (a) or (b) in the case of bovine animals aged lessthan 30 monthsintended for meat production which:
— come from a beef holding officially tuberculosis-free and offi-
cially brucellosis-free,
— are accompanied by an animal health certificate with paragraph
7 in Section A of Annex F Model 1 duly completed,
— remain under supervision until their slaughter,
— have not come into contact during transport with bovine
animalsnot coming from herdsofficially free from thos e
diseases,
and provided that:
— these arrangements are restricted to trade between Member
States or regions of Member States with the same health statuswith regard to tuberculosis or brucellosis,
— the Member State of destination takes all necessary measures to
avoid any contamination of indigenousherds ,
— the Member States put in place a proper system of random
sampling, inspections and controls designed to ensure the effi-cient implementation of these rules,
— the Commission monitors the proper operation of this Directive
so as to ensure that Member States comply fully with the rules;
3. Bovine animals for slaughter must, in addition to the require-
mentsin Articles3, 4 and 5, come from herdsthat are officially
tuberculosis free, officially enzootic-bovine-leukosis free and in thecase of uncastrated bovines, from herds that are officially brucellosisfree.
However, until ►
M4631 December 2000 ◄, the destination coun-
triesmay grant to Spain general or limited licencesto introduce intotheir territoriesanimalsfor s laughter from herdswhich are not offi-
cially free of tuberculosis, enzootic bovine leukosis and brucellosis,
provided such animals:
— have in the 30 daysprior to embarkation undergone the appropriate
tests laid down in Annexes B, C and D, with negative results,
— are taken on arrival in the country of destination directly to a
slaughterhouse and are slaughtered there as soon as possible but atleast within 72 hours of arrival, in accordance with animal healthrequirements.1964L0432 — EN — 01.05.2004 — 009.001 — 10
▼M46
▼M42
▼M42
Article 7
Animalsfor s laughter which have been taken on arrival in the country
of destination:
— to a slaughterhouse, must be slaughtered there as soon as possible
but at least within 72 hours of arrival, in accordance with animalhealth requirements, or
— to an approved assembly centre, must be removed after the market
directly to a slaughterhouse to be slaughtered as soon as possible
but at the latest within three working days of arrival at the assembly
centre, in accordance with animal health requirements. At no time,between their arrival at the assembly centre and their arrival at theslaughterhouse, may they come into contact with cloven-hoofedanimalsother than animalsthat fulfil the conditionslaid down inthisDirective.
Article 8
Member States shall ensure that the suspected presence of any of the
diseases referred to in Annex E (I) is compulsorily and immediately
notifiable to the competent authority.
Each Member State shall forward to the Commission by 31 May each
year, and for the first time in 1999, details of the occurence of diseaseslisted in Annex E (I) and of any other diseases covered by the addi-tional guaranteesprovided for by Community legis lation in its
territory in the previouscalendar year including detailsof the moni-
toring and eradication programmesin operation. Thisinformationshall be based on uniform criteria to be established by the procedureprovided for in Article 17. The Commission shall present this informa-tion to the Member Statesin the framework of the SVC and in
particular may utilize it in relation to the decisions referred to in
AnnexesA and D.
Article 9
1. A Member State which hasa compuls ory national control
programme for one of the contagious diseases listed in Annex E (II)for all or part of its territory may submit the said programme to theCommission, outlining in particular:
— the distribution of the disease in the Member State,
— the reasons for the programme, taking into consideration the impor-
tance of the disease and the programme's likely benefit in relationto itscos t,
— the geographical area in which the programme will be imple-
mented,
— the status categories to be applied to the animal establishments, the
standards which must be attained in each category, and the testproceduresto be us ed,
— the programme monitoring procedures, the results of which must be
supplied at least annually to the Commission,
— the action to be taken if, for any reason, an establishment loses its
status,
— the measures to be taken if the results of the tests carried out in
accordance with the provisions of the programme are positive.
2. The Commission shall examine the programmes presented by the
Member States. Programmes as referred to in paragraph 1 may beapproved in compliance with the criteria laid down in paragraph 1 inaccordance with the procedure provided for in Article 17. Accordingto the same procedure, the additional guarantees, general or limited,which may be required in intra-Community trade, shall be defined at
the same time or at the latest three months after approval of the
programmes. Such guarantees must not exceed those which theMember State implementsnationally.
3. Programmess ubmitted by Member Statesmay be amended or
supplemented in accordance with the procedure laid down in Article17. Amendmentsor additionsto programmeswhich have already been1964L0432 — EN — 01.05.2004 — 009.001 — 11
▼M42
approved or to guaranteeswhich have been defined in accordance with
paragraph 2 may be approved under the same procedure.
Article 10
1. Where a Member State considers that its territory or part of its
territory is free from one of the diseases listed in Annex E (II), it shallpresent to the Commission appropriate supporting documentation,setting out in particular:
— the nature of the disease and the history of its occurrence in its
territory,
— the results of surveillance testing based on serological, microbiolo-
gical, pathological or epidemiological investigation and on the factthat the disease must by law be notified to the competent authori-ties,
— the period over which the surveillance was carried out,— where applicable, the period during which vaccination against the
disease has been prohibited and the geographical area concernedby the prohibition,
— the arrangements for verifying the absence of the disease.
2. The Commission shall examine documentation submitted by
Member States. The additional guarantees, general or specific, which
may be required in intra-Community trade may be defined in accor-dance with the procedure laid down in Article 17. Such guaranteesmust not exceed those which the Member State implements nationally.
3. The Member State concerned shall notify the Commission of any
change in the detailss pecified in paragraph 1 which relate to thedisease, in particular regarding any new outbreaks of the disease. The
guaranteesdefined in accordance with paragraph 2 may, in the light of
such notification, be amended or withdrawn in accordance with theprocedure laid down in Article 17.
Article 11
1. Member States shall ensure that, in order to be approved by the
competent authority, assembly centres meet the following conditionsat least. They must:
(a) be under the control of an official veterinarian who shall ensure
that, in particular, the provisions of Article 4 (1) and (2) arecomplied with;
(b) be located in an area which is not subject to prohibiiton or restric-
tionsin accordance with relevant Community legis lation and/ornational legislation;
(c) be cleaned and disinfected before use, as required by the official
veterinarian;
(d) they must have, taking into account the animal capacity of the
assembly centre:
— a facility dedicated exclusively for this purpose when used as
an assembly centre,
— appropriate facilitiesfor loading, unloading and adequate
housing of a suitable standard for the animals, for wateringand feeding them, and for giving them any necessary treatment;
these facilities must be easy to clean and disinfect,
— appropriate inspection facilities,
— appropriate isolation facilities,— appropriate equipment for cleaning and disinfecting rooms and
trucks,
— an appropriate storage area for fodder, litter and manure,
— an appropriate system for collecting waste water,— the use of an office for the official veterinarian;
(e) admit only animalsthat are identified and come from herdsthat are
officially free of tuberculosis, brucellosis and leukosis or slaughteranimals meeting the conditions set out in the present Directive and1964L0432 — EN — 01.05.2004 — 009.001 — 12
▼M42
in particular Article 6 (3). To thisend, when animalsare admitted
the owner or person in charge of the centre shall ensure they areproperly identified and accompanied by health documentsor appro-
priate certificatesfor the s peciesand categoriesinvolved;
(f) be regularly inspected in order to ascertain that the requirements
for approval continue to be fulfilled.
2. The owner or person in charge of the assembly centre shall be
required, on the basis either of the accompanying documents for theanimalsor of the identification numbersor marksof the animals , to
record on a register or a data base and retain for a minimum period of
three yearsthe following information:
— the name of the owner, the origin, date of entry and exit, number
and identification of the bovine animalsor the regis tration number
of the holding of origin or of the herd of origin of the pigsentering
the centre and their proposed destination,
— the registration number of the transporter and the licence number of
the lorry delivering or collecting animalsfrom the centre.
3. The competent authority shall issue an approval number to each
approved assembly centre. Such approval may be limited to a particulars peciesor to animalsfor breeding and production or to animalsforslaughter. The competent authority shall notify the Commission of thelist of approved assembly centres and of any updates. The Commissionshall present this information to Member States in the framework of the
SVC.
4. The competent authority may suspend or withdraw approval in
the event of failure to comply with thisArticle or other appropriateprovisions of this Directive or other directives in respect of healthrestrictions. Approval may be restored when the competent authorityis satisfied that that assembly centre is in full compliance with all the
appropriate provisions of this Directive.
5. The competent authority shall ensure that when operating
assembly centres have sufficient approved veterinarians to carry out
all duties.
6. Any detailed rulesrequired for uniform application of thisArticle
shall be adopted in accordance with the procedure provided for inArticle 17.
Article 12
1. Member States shall ensure that the transporters referred to in
Article 5 of Directive 91/628/EEC meet the following additional condi-tions:
(a) for the carriage of animals they must use means of transport that
are:
— constructed in such a way that the animal faeces, litter or feed
cannot leak or fall out of the vehicle,
— cleaned and disinfected immediately after every animal trans-
port operation or that of any product which could affectanimal health and if necessary before any new loading ofanimals, using disinfectants officially authorized by the compe-tent authority;
(b) they must either have appropriate cleaning and disinfection facil-
itiesapproved by the competent authority, including facilitiesforstoring litter and dung, or they must provide documentary evidencethat these operations are perfomed by a third party approved by thecompetent authority.
2. The transporter must ensure that for each vehicle used for the
transport of animals a register is kept containing the following informa-1964L0432 — EN — 01.05.2004 — 009.001 — 13
▼M42
tion at least which shall be retained for a minimum period of three
years:
(i) places and dates of pick-up, and the name or business name and
address of the holding or assembly centre where the animals arepicked up;
(ii) places and dates of delivery, and the name or business name and
address of the consignee(s);
(iii) species and number of animals carried;
(iv) date and place of disinfection;
(v) detailsof accompanying documentation (s erial number, etc.).
3. Transporters shall ensure that the consignment of animals do not
at any time, between leaving the holdings or the assembly centre oforigin and arriving at their destination, come into contact with animalsof a lower health status.
4. Member States shall ensure that transporters give a written under-
taking stating in particular that:
— all the measures necessary to comply with this Directive shall be
taken and in particular the provisions laid down in this Article and
relating to the appropriate documentation that must accompany theanimals,
— the transport of animals is entrusted to staff who possess the neces-
sary ability, professional competence and knowledge.
5. In the event of failure to comply with the provisions of this
Article the requirementsof Article 18 of Directive 91/628/EEC s hall
applymutatis mutandis relating to animal health.
Article 13
1. Member States shall ensure that all dealers are registered,
approved and issued with an approval number by the competentauthority and that they comply with the following conditionsat leas t:
(a) they must deal only in animals that are identified and come from
herds that are officially free of tuberculosis, brucellosis andleukosis or slaughter animals meeting the conditions set out in thisDirective and in particular Article 6 (3). To thisend, the dealershall ensure that the animals are properly identified and are accom-panied by health documentsasappropriate for the s peciesinvolved.
However, the competent authority may authorize the marketing of
identified animalswhich do not fulfil the conditionslaid down inthe first paragraph, in so far as they are brought direct to a slaugh-
terhouse in the Member State of origin without passing through
their facilities, for slaughter as soon as possible in order to preventthe spreading of diseases. The necessary provisions should be takento ensure that such animals, when they reach the abattoir, cannotcome into contact with other animalsand that they are s laughtered
apart from other animals;
(b) the dealer shall be required, either on the basis of the document
accompanying the animals, or on the basis of identification
numbers or marks on the animals, to keep a record or data base
and to store the following data for at least three years:
— the name of the owner, origin, date of purchase, categories,
number and identification of bovine animalsor regis tration
number of the holding of origin or of the herd of origin of
pigspurchas ed,
— the registration number of the transporter and/or the licence
number of the lorry delivering and collecting animals,
— the name and address of the purchaser and the destination of the
animals,
— copiesof route plansand/or s erial number of health certificates
asapplicable;1964L0432 — EN — 01.05.2004 — 009.001 — 14
▼M42
(c) when the dealer keeps animals on his premises he shall ensure that:
— specific training is given to the staff in charge of the animals in
applying the requirementsof thisDirective and in the care andwelfare of the animals,
— controls and tests if necessary on the animals are carried out
regularly by the official veterinarian and that all necessary stepsare taken to prevent the spread of disease.
2. Member States shall ensure that all premises used by a dealer in
connection with his business are registered and issued with an approval
number by the competent authority and that they comply with the
following conditionsat leas t:
(a) they must be under the control of an official veterinarian;
(b) they must be located in an area which is not subject to prohibition
or restrictions in accordance with the relevant Community legisla-
tion or national legislation;
(c) they must have:
— appropriate facilitiesof s ufficient capacity and in particular
inspection facilities and isolation facilities so that all animalscan be isolated in the event of an outbreak of a contagiousdisease,
— appropriate facilities for unloading and where necessary
adequate housing of a suitable standard for the animals, forwatering and feeding them, and for giving them any necessary
treatment; these facilities must be easy to clean and disinfect,
— an appropriate reception area for litter and manure,
— an appropriate system for collecting waste water;
(d) be cleaned and disinfected before use, as required by the official
veterinarian.
3. The competent authority may suspend or withdraw approval in
the event of failure to comply with thisArticle or other appropriate
provisions of this Directive or other directives in respect of health
restrictions. Approval may be restored when the competent authorityis satisfied that the dealer is in full compliance with all the appropriateprovisions of this Directive.
4. The competent authority must carry out regular inspections in
order to ascertain that the requirements of this Article are fulfilled.
Article 14
1. The competent authority in a Member State may introduce a
system of surveillance networks.
The surveillance network system must comprise at least the following
elements:
— the herds,
— the owner or any other natural or legal person responsible for the
holding,
— the approved veterinarian or the official veterinarian responsible for
the holding,
— the official veterinary service of the Member State,— the official veterinary diagnostic laboratories or any other labora-
tory approved by the competent authority,
— a computer database.
Official veterinarians for the slaughtering establishments and approved
assembly centres will be associated with the network system.
2. The main objectives of the surveillance network system are to
make the official classification of holdings, to maintain such classifica-
tion by regular inspection, to collect epidemiological data and to carryout disease monitoring so as to ensure compliance with all the provi-sions of this Directive and other directives in respect of healthrestrictions.1964L0432 — EN — 01.05.2004 — 009.001 — 15
▼M42
This surveillance network system shall be mandatory on all holdings in
the territory of the Member State operating such a system. However,the competent authority may authorize the establishment of such a
network on part of the territory made up of one or several adjacent
regionsasdefined in Article 2 (2) (p). Where thisderogation isaccorded, animal movementsto that part of the territory from otherregions which are not part of the network system shall be subject tothe provisions of this Directive.
The competent authority shall lay down the obligations and rights
incumbent upon the approved veterinarians, the persons responsiblefor the holdingsor their ownersand any other participantsin thesystem including the persons responsible for issuing health certificates.
3. The competent authority shall ensure that the obligations referred
to in paragraph 2 at least include the following:
A. Every owner of or person responsible for a holding must:
(i) secure, by contract or legal instrument, the services of a veter-
inarian approved by the competent authority;
(ii) immediately call in the approved veterinarian for the holding
when he suspects the occurrence of an infectious disease or ofany notifiable disease;
(iii) notify the approved veterinarian of all arrivalsof animalson
hisholding;
(iv) isolate the animals before introducing them into his holding to
enable the approved veterinarian to check, where appropriate
by means of the required tests, whether the holding's statusmay be maintained.
B. The approved veterinariansasprovided for by Article 2 (2) (m)
shall be under the control of the competent authority and mustcomply with the following requirements.
They must:
(i) meet the conditions for pursuing the veterinary profession;
(ii) have no financial interest or family links with the owner of or
person responsible for the holding;
(iii) possess particular knowledge in the field of animal health as it
appliesto animalsof the s peciesconcerned. Thismeansthatthey must:
— regularly update their knowledge, especially as regards the
relevant health regulations,
— meet the requirementslaid down by the competent
authority to ensure the proper functioning of the network,
— provide the owner of or person responsible for the holding
with information and assistance in order that all steps aretaken to ensure that the holding's status is maintained, parti-
cularly on the basis of programmes agreed with the
competent authority,
— ensure compliance with the requirements concerning:
(i) the identification and health certification of the
animalsof the herd, the animalsintroduced and thos etraded;
(ii) compulsory reporting of infectious animal diseases and
any other risk factor for animal health or welfare, and
for human health;
(iii) establishing as far as possible the cause of death of
animalsand where they are to be cons igned;
(iv) the hygiene conditionsof the herd and of the lives tock
production units.
If the proper functioning of the system so requires, each
Member State may limit the veterinarians' responsibility toa specific number of holdings or to a specific geographicalarea.
The competent authority shall draw up lists of approved
veterinariansand of the approved holdingsparticipating in1964L0432 — EN — 01.05.2004 — 009.001 — 16
▼M42
the network. If the competent authority findsthat a partici-
pant in the network no longer fulfilsthe conditionss et outabove, it shall suspend or withdraw approval, without
prejudice to any penaltiesthat may be applied.
C. The computer database must contain at least the following informa-
tion:
(1) For each animal:
— identification code,
— date of birth,
— sex,
— breed or colour of coat,
— identification code of the mother or, in the case of an animal
imported from a third country, the identification number
given following inspection under Directive 92/102/EEC andcorresponding to the identification number of origin,
— identification number of the holding where born,
— identification numbersof all holdingswhere the animal has
been kept and the datesof each change of holding,
— date of death or slaughter.
(2) For each holding:
— an identification number consisting of not more than 12
figures(apart from the country code),
— name and address of the holder.
(3) The database must be able to supply the following particulars at
any time:
— the identification number of all animalsof the bovine
species present on a holding, or in the case of groups ofanimals of the porcine species, the registration number ofthe holding of origin or herd of origin and the number ofthe health certificate where applicable,
— a list of all changes of holding for each animal of the bovine
species starting from the holding of birth, or the holding ofimportation in the case of animals imported from third coun-
tries; and for groups of pigs the registration number of the
last holding or last herd and for imported animals from thirdcountriesthe holding of importation.
These particulars will be held on the database until three conse-
cutive years have elapsed since the death of the bovine animalor until three consecutive years have elapsed since the record
was made in the case of records for pigs.
However, only points2, 3 and 4 s hall be applicable to porcine
animals.
(4) In order to ensure the operation of the national computer data-
bases concerning porcine animals, appropriate rules of
application, including the information that the national data-
bases must contain, shall be adopted in accordance with theprocedure laid down in Article 17.
4. All participants in the surveillance network other than those
provided for in 3A and B shall be accountable to the competent
authority. The competent authority in each Member State shall beresponsible for setting up the network and shall carry out regularchecksto ens ure that it operatesproperly.
5. Member States which introduce a system of surveillance networks
asoutlined in paragraphs1 to 4, operational for a period of at leas t 12months, shall apply to the Commission to have it approved under theprocedure provided for in Article 17.
For this purpose the Commission shall examine documentation
submitted by Member States.1964L0432 — EN — 01.05.2004 — 009.001 — 17
▼M45
▼M42
▼M42
The Commission experts shall validate the systems by means of a
system of audits. Where the result of the audit is favourable theCommission shall within 90 days of receipt of the request for approval
make a report to the SVC together with appropriate proposals.
Where repeated offencesare noted, the approval of the s urveillance
network system can be suspended according to the procedure laiddown in Article 17, at the request of the Commission or of one or
more Member States.
6. Member Stateswhich have implemented in all their territory a
recognized surveillance network system as laid down in this Articleshall be authorized not to apply the provision referred to in Article 3(2) (a), second indent to animal movements referred to by this Direc-
tive within their own territory.
7. Not later than 31 December 1999, acting on the basis of a report
from the Commission, accompanied by proposals on which it shall actby a qualified majority, the Council shall review the provisions of thisArticle in the light of experience with a view to amending and updating
them and, if appropriate, extending them to all Member States.
8. The financing of the surveillance network system will be covered
within the framework of the revision of Annex B to Directive 85/73/EEC (
1) in accordance with the provisions laid down in Article 8 of
Directive 96/43/EC.
Article 15
1. Member States shall take the appropriate specific measures to
penalize any infringement of thisDirective whether by a natural or alegal person.
2. If it isconfirmed that the provis ionsof thisDirective are not or
have not been complied with the competent authority of the place in
which such a finding is made shall take all appropriate measures tosafeguard animal health and to prevent the spread of disease.
Depending on the circumstances, such action by the competent
authority may consist of taking the measures required to:
(a) arrange for the journey to be completed or the animalsreturned to
their place of departure by the most direct route, providing thiscourse of action would not further jeopardize the health or welfareof the animals;
(b) arrange for the animalsto be held in s uitable accommodation with
appropriate care in the event of interruption of the journey;
(c) arrange for the slaughter of the animals. The destination and use of
such animals after slaughter shall be regulated:
— in accordance with the provisions of Directive 64/433/EEC (
2),
or
— in accordance with the provisions of Directive 90/667/EEC
where the health status of the animals cannot be established orwhere they are liable to be a risk to animal health or public
health. However, where the provisions of Directive 90/667/
EEC should apply, a regularization period may be accorded tothe owner or his agent before that final possibility is invoked.In that case, the provisions of paragraph 3 of this Article shallapply.
3. The competent authority of the Member State of destination shall
immediately notify the competent authority of the Member State oforigin on the establishment of any infringement of this Directive.1964L0432 — EN — 01.05.2004 — 009.001 — 18
(1) OJ No L 32, 5. 2. 1985, p. 14. Directive asamended by Directive 96/43/EC
(OJ No L 162, 1. 7. 1996, p. 1).
(2) OJ No 121, 29. 7. 1964, p. 2012/64. Directive aslas t amended by Directive
95/23/EC (OJ No L 243, 11. 10. 1995, p. 7).
▼M42
In accordance with the provisions established by Directive 89/608/
EEC, Member States shall provide mutual assistance to one another inthe application of thisDirective in order to ens ure, in particular,
compliance with the provisions laid down in this Article.
4. ThisArticle s hall not affect national rulesapplicable to penal
sanctions.
Article 16
1. AnnexesA, D (Chapter I), E and F s hall be amended by the
Council, acting by a qualified majority on a Commission proposal, in
particular with regard to their adaptation to technological and scientific
developments.
Annexes B, C and D (Chapter II) shall be amended by the Commission
in accordance with the proceduresprovided for in Article 17.
However,
(a) before 1 July 1997, the Commission shall submit to the Council a
proposal for the amendment of Annexes A and D (Chapter I), for
the purpose of updating them and if necessary it will apply thesame procedure to Annex F. The Council shall decide on theseproposals by a qualified majority before 1 January 1998;
(b) before 30 June 1998, the Commission, in accordance with the
procedure laid down in Article 17 and on the basis of the opinionof the Scientific Veterinary Committee, shall update and if neces-
sary amend Annexes B, C and D (Chapter II) to adapt them to
scientific developments.
2. Not later than 31 December 1999, acting on the basis of a report
from the Commission, accompanied by suitable proposals on which it
shall act by a qualified majority, the Council shall review the provi-
sions of this Directive in the light of experience with a view toamending and updating them so that they comply with the rules laiddown for completion of the internal market.
3. Where necessary to facilitate the changeover to the new arrange-
ments provided for in this Directive, the Commission, acting in
accordance with the procedure laid down in Article 17a, may adopttransitional measures applicable for a period of not more than twoyears.
Article 17
1. The Commission shall be assisted by the Standing Veterinary
Committee established by Decision 68/361/EEC (hereinafter referredto asthe ‘Committee’).
2. Where reference ismade to thisparagraph, Articles5 and 7 of
Decision 1999/468/EC shall apply.
The period laid down in Article 5(6) of Decision 1999/468/EC shall be
set at three months.
3. The Committee shall adopt its rules of procedure.
Article 17a
1. The Commission shall be assisted by the Standing Veterinary
Committee established by Decision 68/361/EEC (hereinafter referredto asthe ‘Committee’).
2. Where reference ismade to thisparagraph, Articles5 and 7 of
Decision 1999/468/EC shall apply, having regard to the provisions ofArticle 8 thereof.
The period laid down in Article 5(6) of Decision 1999/468/EC shall be
set at three months.
3. The Committee shall adopt its rules of procedure.1964L0432 — EN — 01.05.2004 — 009.001 — 19
▼M46
▼M45
Article 18
Those Member States which have not introduced an approved surveil-
lance network system shall ensure that a computer database complying
with the provisions laid down in Article 14 is fully operational as
follows:
(a) for bovine animals, from 31 December 1999;
(b) for a register of holdings of porcine animals, complying with the
provisions laid down in Article 14(3)(c)(2) from 31 December2000;
(c) for movements of porcine animals, complying with the provisions
laid down in Article 14(3)(c)(3):
— from their holding of birth, by 31 December 2001,
— from all other holdings, by 31 December 2002.
There shall be an entry in the database for each separate movement of
porcine animals. The entry shall comprise at least the following: the
number of porcine animalsbeing moved, the identification number of
the holding or herd of departure, the identification number of theholding or herd of arrival and the departure and arrival dates.
Article 19
The ruleslaid down in Directive 90/425/EEC s hall apply in particular
to checksat origin, to the organization of, and follow-up to, the checksto be carried out by the country of destination, and to the safeguard
measures to be implemented.
Article 20
This Directive is addressed to the Member States.1964L0432 — EN — 01.05.2004 — 009.001 — 20
▼M42
▼M44
ANNEX A
I.Officially tuberculosis-freebovineherd
For the purposes of this section ‘bovine animals’ means all bovine animals
with the exception of animals taking part in cultural or sporting events.
1. A bovine herd is officially tuberculosis-free if:
(a) all the animals are free from clinical signs of tuberculosis;
(b) all the bovine animalsover s ix weeksold have reacted negatively to
at least two official intradermal tuberculin tests carried out in accor-
dance with Annex B, the first six months after the elimination of
any infection from the herd and the second six months later or,
where the herd has been assembled solely from animals that origi-
nate in officially tuberculosis-free herds, the first test shall be
carried out at least 60 days after assembly and the second shall not
be required;
(c) following the completion of the first test referred to in (b), no
bovine animal over six weeks old has been introduced into the
herd unless it has reacted negatively to an intradermal tuberculin
test performed and assessed according to Annex B and carried out
either in the 30 daysprior to, or the 30 daysafter the date of its
introduction into the herd; in the latter case the animal(s) must be
isolated physically from the other animals of the herd in a way to
avoid any direct or indirect contact with the other animalsuntil
proven negative.
However, the competent authority may not require thistes t to be
carried out for movementsof animalson itsown territory if the
animal is from an officially tuberculosis-free herd, except in a
Member State where, on 1 January 1998 and until the status of offi-
cially tuberculosis-free region is obtained, the competent authority
required such tests to be carried out for animals moving between
herds participating in a network system as referred to in Article 14.
2. A bovine herd will retain officially tuberculosis-free status if:
(a) the conditionsdetailed in 1(a) and (c) continue to apply;
(b) all animalsentering the holding come from herdsof officially tuber-
culosis-free status;
(c) all animalson the holding, with the exception of calvesunder s ix
weeksold which were born in the holding, are s ubjected to routine
tuberculin testing in accordance with Annex B at yearly intervals.
However, the competent authority of a Member State may, for the
Member State or part of the Member State where all the bovine
herds are subject to an official programme to combat tuberculosis,
alter the frequency of the routine tests as follows:
— if the average — determined at 31 December of each year — of
the annual percentagesof bovine herdsconfirmed asinfected
with tuberculosis is not more than 1 % of all herds within the
defined area during the two most recent annual supervisory
periods, the interval between routine herd tests may be increased
to two yearsand male animalsfor fattening within an is olated
epidemiological unit may be exempted from tuberculin testing
provided that they come from officially tuberculosis-free herds
and that the competent authority guaranteesthat the malesfor
fattening will not be used for breeding and will go direct for
slaughter,
— if the average — determined at 31 December of each year — of
the annual percentagesof bovine herdsconfirmed asinfected
with tuberculosis is not more than 0,2 % of all herds within the
defined area during the two most recent biennial supervisory
periods, the interval between routine tests may be increased to
three yearsand/or the age at which animalshave to undergo
these tests may be increased to 24 months,
— if the average — determined at 31 December of each year — of
the annual percentagesof bovine herdsconfirmed asinfected
with tuberculosis is not more than 0,1 % of all herds within the
defined area during the two most recent supervisory triennial
periods, the interval between routine tests may be increased to
four years, or, providing the following conditions are met, the1964L0432 — EN — 01.05.2004 — 009.001 — 21
▼M44
competent authority may dispense with tuberculin testing of the
herds:
(1) before the introduction into the herd all the bovine animals
are subjected to an intradermal tuberculin test with negative
results;
or
(2) all bovine animals slaughtered are examined for lesions of
tuberculosis and any such lesions are submitted to a histo-
pathological and bacteriological examination for evidence
of tuberculosis.
The competent authority may also, in respect of the Member State
or a part thereof, increase the frequency of tuberculin testing if the
level of the disease has increased.
3A. The officially tuberculosis-free status of a herd is to be suspended if:
(a) the conditionsdetailed in paragraph 2 are no longer fulfilled;
or
(b) one or more animalsare deemed to have given a pos itive reaction
to a tuberculin test, or a case of tuberculosis is suspected at post-
mortem examination.
When an animal is considered to be a positive reactor it will be
removed from the herd and slaughtered. Appropriate post-mortem,
laboratory and epidemiological examinationss hall be carried out
on the positive reactor or the carcase of the suspect animal. The
status of the herd will remain suspended until such time as all
laboratory examinationshave been completed. If the pres ence of
tuberculosis is not confirmed, the suspension of the officially tuber-
culosis-free status may be lifted following a test of all animals over
six weeks of age with negative results at least 42 days after the
removal of the reactor animal(s);
or
(c) the herd contains animals of unresolved status as described in
Annex B. In this case, the status of the herd is to remain suspended
until the animals' status has been clarified. Such animals must be
isolated from the other animals of the herd until their status has
been clarified, either by a further test after 42 days or by post-
mortem and laboratory examination;
(d) however, by way of derogation from the requirementsof paragraph
(c), in a Member State where the competent authority carriesout
routine herd testing using the comparative tuberculin test described
in Annex B, and in the case of a herd where no confirmed reactor
animals have been disclosed for at least three years, the competent
authority may decide not to restrict the movement of other animals
in the herd, provided that the status of any inconclusive reactors is
resolved by a further test after 42 days and that no animals from the
holding are allowed to enter into intra-Community trade until the
status of any inconclusive reactors has been resolved. If at this
further test any animal either gives a positive reaction or continues
to give an inconclusive reaction, then the conditions of paragraph(b) apply. If the presence of disease is subsequently confirmed, all
animals leaving the holding since the time of the last clear herd test
must be traced and tested.
3B. The officially tuberculosis-free status of the herd is to be withdrawn if
the presence of tuberculosis is confirmed by the isolation of M. bovis on
laboratory examination.
The competent authority may withdraw status if:
(a) the conditionsdetailed in point 2 are no longer fulfilled, or
(b) classical lesions of tuberculosis are seen at post-mortem examina-
tion, or
(c) an epidemiological enquiry establishes the likelihood of infection,
(d) or for any other reasons considered necessary for the purpose of
controlling bovine tuberculosis.
Tracing and checking isto be undertaken by the competent authority of
any herd considered to be epidemiologically related. The officially
tuberculosis-free status of a herd is to remain withdrawn until cleansing1964L0432 — EN — 01.05.2004 — 009.001 — 22
▼M46
▼M44
▼M44
and disinfection of the premises and utensils has been completed and all
animals over six weeks of age have reacted negatively to at least two
consecutive tuberculin tests, the first no less than 60 days and the
second no less than four months and no more than 12 months after the
removal of the last positive reactor.
4. On the basis of information supplied in accordance with Article 8, a
Member State or part of a Member State may be declared officially
tuberculosis-free according to the procedure laid down in Article 17 if
it meetsthe following conditions :
(a) the percentage of bovine herdsconfirmed asinfected with tubercu-
losis has not exceeded 0,1 % per year of all herds for six
consecutive years and at least 99,9 % of herds have achieved offi-
cially tuberculosis-free status each year for six consecutive years,
the calculation of thislatter percentage to take place on 31
December each calendar year;
(b) each bovine animal isidentified in accordance with Community
legislation;
(c) all bovine animals slaughtered are subjected to an official post-
mortem examination;
(d) the procedures for suspension and withdrawal of officially tubercu-
losis-free status are complied with.
5. The Member State or part of a Member State will retain officially tuber-
culosis-free status if the conditions 4(a) to (d) continue to be met.
However, if there is evidence of a significant change in the situation
asregardstuberculos isin a Member State or part of a Member State
which has been recognised as officially tuberculosis-free, the Commis-
sion may, in accordance with the procedure laid down in Article 17,
take a Decision suspending or revoking the status until the requirements
of the Decision have been fulfilled.
II.Officially brucellosis-free andbrucellosis-free bovineherds
For the purposes of this section ‘bovine animals’ means all bovine animals
with the exception of malesfor fattening provided that they come from offi-
cially brucellosis-free herds and that the competent authority guarantees that
the malesfor fattening will not be us ed for breeding and will go direct for
slaughter.
1. A bovine herd is officially brucellosis-free if:
(a) it containsno bovine animalswhich have been vaccinated agains t
brucellosis, except females which have been vaccinated at least
three yearsprevious ly;
(b) all the bovine animalshave been free from clinical s ignsof brucel-
losis for at least six months;
(c) all the bovine animalsover 12 monthsold have been s ubjected to
one of the following test regimes with negative results in accor-
dance with Annex C:
(i) two serological tests specified in paragraph 10 at an interval of
more than three months and less than 12 months;
(ii) three tests on milk samples at three-monthly intervals followed
at least six weeks later by a serological test specified in para-
graph 10;
(d) any bovine animal entering the herd comesfrom a herd of officially
brucellosis-free status and, in the case of bovine animals over 12
months old, has shown a brucella titre of less than 30 IU of agglu-
tination per ml when given a serum agglutination test in accordance
with Annex C or hasreacted negatively to any other tes t approved
in accordance with the procedure at Article 17 during the 30 days
prior to or the 30 daysafter the date of itsintroduction into the
herd: in the latter case, the animal(s) must be isolated physically
from the other animalsof the herd in s uch a way asto avoid direct
or indirect contact with the other animalsuntil proven negative.
2. A bovine herd will retain officially brucellosis-free status if:
(a) one of the following test regimes is carried out annually with nega-
tive results in accordance with Annex C:
(i) three milk ring tests carried out at intervals of at least three
months;1964L0432 — EN — 01.05.2004 — 009.001 — 23
▼M46
▼M44
▼M44
(ii) three milk ELISAscarried out at intervalsof at leas t three
months;
(iii) two milk ring tests carried out at an interval of at least three
months followed at least six weeks later by a serological test
referred to in paragraph 10;
(iv) two milk ELISAscarried out at an interval of at leas t three
months followed at least six weeks later by a serological test
referred to in paragraph 10;
(v) two serological tests carried out at an interval of at least three
monthsand not more than 12 months .
However, the competent authority of a Member State may, for the
Member State or part of the Member State which isnot officially
brucellosis-free but where all the bovine herds are subject to an
official programme to combat brucellosis, alter the frequency of
the routine tests as follows:
— where not more than 1 % of bovine herdsare infected, it may be
sufficient to carry out each year two milk ring tests or two milk
ELISAs at an interval of at least three months, or one serolo-
gical test,
— where at least 99,8 % of bovine herds have been recognised as
officially brucellosis-free for at least four years, the interval
between checksmay be extended to two yearsif all animals
over 12 months of age are tested, or testing may be restricted
to animalsover 24 monthsof age if herdscontinue to be tes ted
each year. The checks must be carried out using one of the sero-
logical tests referred to in paragraph 10;
(b) all bovine animalsentering the herd come from herdsof officially
brucellosis-free status and, in the case of bovine animals over 12
months old, have shown a brucella titre of less than 30 IU of agglu-
tination per ml when given a serum agglutination test in accordance
with Annex C or have reacted negatively to any other test approved
in accordance with the procedure at Article 17 during the 30 days
prior to or the 30 daysafter the date of their introduction into the
herd; in the latter case, the animal(s) must be isolated physically
from the other animalsof the herd in s uch a way asto avoid direct
or indirect contact with the other animalsuntil proven negative.
However, the test described in point (b) need not be required in
Member States, or regions of Member States, where the percentage
of bovine herdsinfected with brucellos ishasnot exceeded 0,2 %
for at least two years and where the animal comes from an offi-
cially brucellosis-free bovine herd within that Member State or
region and hasnot during trans portation come into contact with
bovine animals of lesser status;
(c) notwithstanding point (b), bovine animals from a brucellosis-free
bovine herd may be introduced into an officially brucellosis-free
herd if they are at least 18 months old and, if vaccinated against
brucellosis, the vaccination was carried out more than a year
previously.
Such animals must have shown, in the 30 days prior to introduction,
a brucella titre lower than 30 IU of agglutination per ml and a nega-
tive result when given a complement fixation test, or other test
approved under the procedure set out in Article 17.
If, however, a female bovine animal from a brucellosis-free herd is
introduced into an officially brucellosis-free herd, under the provi-
sions of the above paragraph, that herd shall be considered to be
brucellosis-free for two years from the date on which the last vacci-
nated animal wasintroduced.
3A. The officially brucellosis-free status of a herd is to be suspended if:
(a) the conditionsdetailed in paragraphs1 and 2 are no longer fulfilled;
or
(b) as a result of laboratory tests or on clinical grounds one or more
bovine animals is suspected of having brucellosis and the suspect
animals have been slaughtered or isolated in a way to avoid any
direct or indirect contact with the other animals.
Where the animal hasbeen s laughtered and isno longer available for
testing, the suspension may be lifted if two serum agglutination tests,
carried out in accordance with Annex C on all bovine animalsin the
herd over 12 monthsold, s how a titre lower than 30 IU of agglutination
per ml. The first test shall be carried out at least 30 days after the
removal of the animal and the second at least 60 days later.1964L0432 — EN — 01.05.2004 — 009.001 — 24
▼M44
Where the animal hasbeen is olated from the animalsin the herd, it may
be reintroduced into the herd and the status of the herd may be restored
following:
(a) a serum agglutination test which has shown a titre lower than 30 IU
of agglutination per ml and hasgiven a negative res ult to a comple-
ment fixation test, or
(b) a negative result to any other combination of tests approved for that
purpose under the procedure set out in Article 17.
3B. The officially brucellosis-free status of the herd is to be withdrawn if, as
a result of laboratory tests or epidemiological investigations, brucella
infection hasbeen confirmed in the herd.
The status of the herd is not to be restored until either all bovine
animalspres ent in the herd at the time of the outbreak have been
slaughtered, or the herd has been subject to check testing and all
animalsover 12 monthsof age have given negative res ultsto two
consecutive tests at 60-day intervals, the first being carried out not less
than 30 days after removal of the positive animal(s).
In the case of bovine animals which were pregnant at the time of the
outbreak, the final check must be carried out at least 21 days after the
last animal pregnant at the time of the outbreak has calved.
4. A bovine herd is brucellosis-free if it complies with the conditions in
1(b) and (c) and when vaccination hasbeen carried out asfollows :
(i) female bovine animalshave been vaccinated:
— before the age of six months old with live strain 19 vaccine, or
— before the age of 15 monthsold with killed 45/20 adjuvant
vaccine which hasbeen officially ins pected and approved, or
— with other vaccinesapproved under the procedure laid down in
Article 17;
(ii) bovine animalsunder 30 monthsold which have been vaccinated
with live strain 19 vaccine may give a serum agglutination test
result greater than 30 IU but less than 80 IU of agglutination per
millilitre provided that, on the complement fixation test, they give
a result less than 30 EEC units in the case of females vaccinated
less than 12 months previously or less than 20 EEC units in all
other cases.
5. A bovine herd will retain brucellosis-free status if:
(i) it is subject to one of the testing regimes listed in 2(a);
(ii) bovine animalsentering the herd comply with the requirementsof
2(b); or
— come from herds of brucellosis-free status, and in the case of
bovine animalsover 12 monthsold, have s hown, in the 30
daysprior to or in is olation after introduction into the herd,
less than 30 IU of agglutination per ml when given a serum
agglutination test and a negative result to a complement fixation
test in accordance with Annex C, or
— come from herds of brucellosis-free status, are under 30 months
old and have been vaccinated with live strain 19 vaccine if they
give a serum agglutination test result greater than 30 IU but less
than 80 IU of agglutination per millilitre provided that, on the
complement fixation test, they give a result less than 30 EEC
units in the case of females vaccinated less than 12 months
previously or less than 20 EEC units in all other cases.
6A. The brucellosis-free status of a herd is to be suspended if:
(a) the conditionsdetailed in paragraphs4 and 5 have not been
complied with; or
(b) as a result of laboratory tests or on clinical grounds one or more
bovine animals over 30 months old is suspected of having brucel-
losis and the animal(s) under suspicion have been slaughtered, or
isolated in a way to avoid any direct or indirect contact with other
animals.
Where the animal hasbeen is olated, it may be reintroduced into the
herd and the status of the herd may be restored, if it subsequently shows
a serum agglutination titre lower than 30 IU of agglutination per ml and
has given a negative result to a complement fixation test, or other test
approved under the procedure set out in Article 17.
Where the animalshave been s laughtered and are no longer available
for testing, the suspension may be lifted if two serum agglutination1964L0432 — EN — 01.05.2004 — 009.001 — 25
▼M44
tests, carried out in accordance with Annex C on all bovine animals in
the holding over 12 monthsold, s how a titre lower than 30 IU of agglu-
tination per ml. The first test is to be carried out at least 30 days after
the removal of the animal and the second at least 60 days later.
If the animals to be tested in the previous two subparagraphs are under
30 monthsold and have been vaccinated with live s train 19 vaccine
they may be considered to be negative if they give a serum agglutina-
tion test result greater than 30 IU but less than 80 IU of agglutination
per millilitre provided that, on the complement fixation test, they give a
result less than 30 EEC units in the case of females vaccinated less than
12 months previously or less than 20 EEC units in all other cases.
6B. The brucellosis-free status of the herd is to be withdrawn if, as a result
of laboratory tests of epidemiological investigations, brucella infection
hasbeen confirmed in a herd. The s tatusof the herd isnot to be
restored until either all the bovine animals present in the herd at the
time of the outbreak have been slaughtered or the herd has been subject
to check testing and all unvaccinated animals over 12 months of age
have given negative results to two consecutive tests at 60 day intervals,
the first being carried out not less than 30 days after removal of the
positive animal(s).
If all the animalsto be tes ted referred to in the preceding paragraph are
less than 30 months old and have been vaccinated with live strain 19
vaccine, they may be considered negative if they show a brucella titre
of more than 30 IU but less than 80 IU of agglutination per ml,
provided that in the complement fixation test they show a titre of less
than 30 EEC units in the case of females vaccinated less than 12
months previously or a titre of less than 20 EEC units in all other cases.
In the case of bovine animals which were pregnant at the time of the
outbreak, the final check must have been carried out at least 21 days
after the last animal pregnant at the time of the outbreak has calved.
7. A Member State or a region of a Member State may be declared offi-
cially brucellosis-free according to the procedure laid down in Article
17 if it meetsthe following conditions :
(a) no case of abortion due to brucella infection and no isolation of B.
abortus has been recorded for at least three years and at least 99,8 %
of herds have achieved officially brucellosis-free status each year
for five consecutive years, the calculation of this percentage to
take place on 31 December each calendar year. However, where
the competent authority adoptsa policy of whole herd s laughter,
isolated incidents shown by epidemiological enquiry to be due to
the introduction of animalsfrom outs ide the Member State or part
of the Member State and herds whose officially brucellosis-free
status has been suspended or withdrawn for reasons other than
suspicion of disease, is to be disregarded for the purpose of the
above calculation provided that the central competent authority of
the Member State concerned by these incidents makes an annual
record and forwards them to the Commission in accordance with
Article 8(2), and
(b) each bovine animal isidentified in accordance with Community
legislation, and
(c) notification of cases of abortion is mandatory and they are investi-
gated by the competent authority.
8. Subject to paragraph 9, a Member State or a region of a Member State
declared officially brucellosis-free is to retain this status if:
(a) the conditions imposed by paragraph 7(a) and (b) are still fulfilled
and notification of cases of abortion suspected of being due to
brucellosis is mandatory and are investigated by the competent
authority;
(b) every year for the first five years after attaining status, all bovine
animalsover 24 monthsof age in not les sthan 20 % of herds
have been tested and have reacted negatively to a serological test
carried out in accordance with Annex C or, in the case of dairy
herds, by examination of milk samples in accordance with Annex
C;
(c) every bovine animal suspected of being infected with brucellosis is
notified to the competent authority and undergoesofficial epidemio-
logical investigation for brucellosis comprising at least two1964L0432 — EN — 01.05.2004 — 009.001 — 26
▼M46
▼M44
▼M44
serological blood tests, including the complement fixation test, and
a microbiological examination of appropriate samples;
(d) during the period of suspicion, which is to continue until negative
results have been obtained from the tests provided for in (c), the
officially brucellosis-free status of the herd of origin or transit of
the suspected bovine animal and of the herds linked epidemiologi-
cally to it is to be suspended;
(e) in the event of an outbreak of brucellosis that has spread, all bovine
animals have been slaughtered. Animals of the remaining suscep-
tible species will undergo appropriate tests and premises and
equipment will be cleaned and disinfected.
9. A Member State or a region of a Member State declared officially
brucellosis-free is to report the occurrence of all cases of brucellosis to
the Commission. If there is evidence of a significant change in the
situation as regards brucellosis in a Member State or part of a Member
State which has been recognised as officially brucellosis-free, the
Commission may according to the procedure laid down in Article 17
propose that the status be suspended or revoked until the requirements
of the Decision have been fulfilled.
10. For the purposes of section II, a serological test means either a serum
agglutination test, buffered brucella antigen test, complement fixation
test, plasma agglutination test, plasma ring test, micro-agglutination
test or individual blood ELISA, as described in Annex C. Any other
diagnostic test approved under the procedure laid down in Article 17
and described in Annex C will also be accepted for the purposes of
section II. A milk test means a milk ring test or a milk ELISA in accor-
dance with Annex C.1964L0432 — EN — 01.05.2004 — 009.001 — 27
▼M49
ANNEX B
TUBERCULOSIS
1. IDENTIFICATION OF THE AGENT
The presence of Mycobacterium bovis (M. bovis) , agent of bovine
tuberculosis, in clinical and post-mortem specimens may be demon-
strated by examination of stained smears or immunoperoxidase
techniquesand confirmed by cultivation of the organis m on primary
isolation medium.
Pathological material for the confirmation of M. bovis should be taken
from abnormal lymph nodesand parenchymatousorganss uch as
lungs, liver, spleen, etc. In the cases where the animal does not
present pathological lesions, samples from the retropharyngeal, bron-
chial, mediastinal, supramammary, mandibular and some mesenteric
lymph nodesand liver s hould be collected for examination and
culture.
Identification of isolates may be usually carried out by determining
cultural and biochemical properties. The polymerase chain reaction
(PCR) may also be employed for the detection of the M. tuberculosis
complex. DNA analysis techniques may prove to be faster and more
reliable than biochemical methodsfor the differentiation of M. bovis
from other membersof the M. tuberculosis complex. Genetic finger-
printing allows distinguishing between different strains of M. bovis
and will enable patterns of origin, transmission and spread of M. bovis
to be described.
The techniques and media used, their standardisation and the interpre-
tation of results must conform to that specified in the OIE Manual of
Standards for Diagnostic Tests and Vaccines, Fourth Edition, 2000,
Chapter 2.3.3 (bovine tuberculosis).
2. THE TUBERCULIN SKIN TEST
Tuberculin PPD (Purified Protein Derivatives) that fulfil the standards
laid down in paragraph 2.1 shall be used for carrying out official
tuberculin skin test following the procedures referred to in paragraph
2.2.
2.1.Standardsfortuberculin(bovineandavian)
2.1.1. Definition
Tuberculin purified protein derivative (tuberculin PPD, bovine or
avian) isa preparation obtained from the heat-treated productsof
growth and lysis of Mycobacterium bovisor Mycobacterium
avium (asappropiate) capable of revealing a delayed
hypersensitivity in an animal sensitised to microorgan-
i sm s o ft h esa m esp e c i e s.
2.1.2. Production
It isobtained from the water-s oluble fractionsprepared by heating in
free-flowing steam and subsequently filtering cultures of M. bovis
or M. avium (asappropiate) grown in a liquid s ynthetic
medium. The active fraction of the filtrate, consisting
mainly of protein, is isolated by precipitation, washed
and re-dissolved. An antimicrobial preservative that does
not give rise to false positive reactions, such as phenol,
may be added. The final sterile preparation, free from
mycobacteria, is distributed aseptically into sterile
tamper-proof glass containers which are then closed so as
to prevent contamination. The preparation may be freeze-
dried.
2.1.3. Identification of the product
Inject a range of graded doses intradermally at different sites into
suitably sensitised albino guinea-pigs, each weighing not less than
250 g. After 24 h to 28 h, reactionsappear in the form of oedematous
swellings with erythema with or without necrosis at the points of
injection. The size and severity of the reactions vary according to
the dose. Unsensitised guinea-pigs show no reactions to similar injec-
tions.1964L0432 — EN — 01.05.2004 — 009.001 — 28
▼M49
2.1.4. Tests
2.1.4.1. pH: The pH is6.5 to 7.5.2.1.4.2. Phenol: If the preparation to be examined containsphenol, itsconcen-
tration isnot more than 5 g/l.
2.1.4.3. Sensitising effect: Use a group of three guinea-pigs that have not been
treated with any material which will interfere with the test. On 3
occasions at intervals of five days inject intradermally into each
guinea-pig a dose of the preparation to be examined equivalent to
500 IU in 0,1 ml. 15 to 21 daysafter the third injection inject the
same dose (500 IU) intradermally into these animals and into a
control group of three guinea-pigs of the same mass and which have
not previously received injections of tuberculin. 24 to 28 hours after
the last injections, the reactions of the two groups are not significantly
different.
2.1.4.4. Toxicity: Use two guinea-pigs, each weighing not less than 250 g and
which have not previously been treated with any material which will
interfere with the test. Inject subcutaneously into each guinea-pig
0,5 ml of the preparation to be examined. Observe the animals for
seven days. No abnormal effects occur during the observation period.
2.1.4.5. Sterility: It complies with the test for sterility prescribed in the mono-
graph on Vaccinesfor veterinary us e 4th Edition 2002 of the
European Pharmacopoeia.
2.1.5. Potency
The potency of tuberculin purified protein derivative (bovine and
avian) is determined by comparing the reactions produced in sensi-
tised guinea-pigs by the intradermal injection of a series of dilutions
of the preparation to be examined with those produced by known
concentrationsof a reference preparation of tuberculin (bovine or
avian, asappropiate) purified protein derivative calibrated in Interna-
tional Units.
To test the potency, sensitise not fewer than nine albino guinea-pigs,
each weighing 400 g to 600 g, by the deep intramuscular injection of
0,0001 mg of wet mass of living M. bovis of strain AN5
suspended in 0.5 ml of a 9 g/l solution of sodium chloride
R for bovine tuberculin, or a suitable dose of inactivated
or live M. avium for avian tuberculin. Not less than four
weeks after the sensitisation of the guinea-pigs, shave
their flanksto provide s pace for not more than four injec-
tion sites on each side. Prepare dilutions of the
preparation to be examined and of the reference prepara-
tion using isotonic phosphate-buffered saline (pH 6,5-
7,5) containing 0,005 g/l of polysorbate 80 R. Use not
fewer than three doses of the reference preparation and
not fewer than three doses of the preparation to be exam-
ined. Choose the doses such that the lesions produced
have a diameter of not less than 8 mm and not more than
25 mm. Allocate the dilutionsrandomly to the s itesus ing
a Latin square design. Inject each dose intradermally in a
constant volume of 0,1 ml or 0,2 ml. Measure the
diametersof the les ionsafter 24 to 28 hoursand calculate
the result of the test using the usual statistical methods
and assuming that the diameters of the lesions are directly
proportional to the logarithm of the concentration of the
tuberculins.
The test is not valid unless the fiducial limits of error (P = 0,95) are
not less than 50 % and not more then 200 % of the estimated potency.
The estimated potency is not less than 66 % and not more than 150 %
of the stated potency for bovine tuberculin. The estimated potency is
not less than 75 % and not more than 133 % of the stated potency for
avian tuberculin. The stated potency is not less than 20 000 IU/ml for
both tuberculins(bovine and avian).
2.1.6. Storage
Store protected from light, at a temperature of 5 ± 3 ºC.
2.1.7. Labelling
The label states:
— the potency in International Unitsper millilitre,
— the name and quantity of any added substance,1964L0432 — EN — 01.05.2004 — 009.001 — 29
▼M49
— for freeze-dried preparations:
— -the name and volume of the reconstituting liquid to be added,
— -that the product should be used immediately after reconstitu-
tion.
2.2.Testprocedures
2.2.1. The following shall be recognised as official intradermal tuberculin
tests:
— the single intradermal test: this test requires a single injection of
bovine tuberculin,
— the intradermal comparative test: this test requires one injection of
bovine tuberculin and one injection of avian tuberculin given
simultaneously.
2.2.2. The dose of tuberculin injected shall be:
— not less than 2 000 IU of bovine tuberculin,
— not less than 2 000 IU of avian tuberculin.
2.2.3. The volume of each injection dose shall not exceed 0,2 ml.2.2.4. Tuberculin tests shall be carried out by injecting tuberculin(s) into the
skin of the neck. The injection sites shall be situated at the border of
the anterior and middle thirdsof the neck. When both avian and
bovine tuberculins are injected in the same animal, the site for injec-
tion of avian tuberculins shall be about 10 cm from the crest of the
neck and the site for the injection of bovine tuberculin about 12,5 cm
lower on a line roughly parallel with the line of the shoulder or on
different sides of the neck; in young animals in which there is not
room to separate the sites sufficiently on one side of the neck, one
injection shall be made on each side of the neck at identical sites in
the centre of the middle third of the neck.
2.2.5. The technique of tuberculin testing and interpretation of reactions
shall be as follows:
2.2.5.1. Technique:
Injection sites shall be clipped and cleansed. A fold of skin within
each clipped area shall be taken between the forefinger and thumb
and measured with callipers and recorded. The dose of tuberculin
shall then be injected by a method that ensures that the tuberculin is
delivered intradermically. A short sterile needle, bevel edge outwards,
with graduated syringe charged with tuberculin, inserted obliquely
into the deeper layers of the skin may be used. A correct injection
shall be confirmed by palpating a small pea-like swelling at each
site of injection. The skin-fold thickness of each injection site shall
be remeasured 72 hours (± 4 hours) after injection and recorded.
2.2.5.2. Interpretation of reactions
The interpretation of reactions shall be based on clinical observations
and the recorded increase(s) in skin-fold thickness at the sites of
injection 72 hoursafter injection of tuberculin(s ).
(a) Negative reaction: if only limited swelling is observed, with an
increase of not more than 2 mm in the thickness of the fold of
skin without clinical signs such as diffuse or extensive oedema,
exudation, necrosis, pain or inflammation of the lymphatic ducts
in that region or of the lymph nodes.
(b) Inconclusive reaction: if no clinical signs such as mentioned in a)
are observed and if the increase in skin-fold thickness is more
than 2 mm and less than 4 mm.
(c) Positive reaction: if clinical signs such as mentioned in a) are
observed or there is an increase of 4 mm or more in the thickness
of the fold of skin at the injection site.
2.2.5.3. The interpretation of official intradermal tuberculin tests shall be as
follows:
2.2.5.3.1. Single intradermal test:
(a) positive: a positive bovine reaction as defined in paragraph
2.2.5.2(c);
(b) inconclusive: an inconclusive reaction as defined in paragraph
2.2.5.2(b);
(c) negative: a negative bovine reaction asdefined in paragraph
2.2.5.2(a).1964L0432 — EN — 01.05.2004 — 009.001 — 30
▼M49
Animals inconclusive to the single intradermal test shall be subjected
to another test after a minimum of 42 days.
Animals which are not negative to this second test shall be deemed to
be positive to the test.
Animals positive to the single intradermal test may be subjected to an
intradermal comparative test if false positive reaction or interference
reaction is suspected.
2.2.5.3.2. Intradermal comparative test for the establishment and maintenance of
officially tuberculosis-free herd status:
(a) positive: a positive bovine reaction which is more than 4 mm
greater than the avian reaction, or the presence of clinical signs;
(b) inconclusive: a positive or inconclusive bovine reaction which is
from 1 to 4 mm greater than the avian reaction, and the absence
of clinical signs;
(c) negative: a negative bovine reaction, or a positive or inconclusive
bovine reaction but which is equal to or less than a positive or
inconclusive avian reaction and the absence of clinical signs in
both cases.
Animals inconclusive to the intradermal comparative test shall be
subjected to another test after a minimum of 42 days. Animals, which
are not negative to this second test, shall be deemed to be positive to
the test.
2.2.5.3.3. Officially tuberculosis-free herd status may be suspended and animals
from the herd shall not be allowed to enter intra-Community trade
until such time as the status of the following animals is resolved:
(a) animals which have been deemed to be inconclusive to the single
intradermal tuberculin test;
(b) animals which have been deemed to be positive to the single
intradermal tuberculin test but are awaiting retest with an intra-
dermal comparative test;
(c) animalswhich have been deemed to be inconclus ive to the intra-
dermal comparative test.
2.2.5.3.4. Where animals are required by Community legislation to be subjected
to an intradermal test prior to movement, the test shall be interpreted
so that no animal which shows an increase in skin-fold thickness
greater than 2 mm or the presence of clinical signs is entered into
intra-Community trade.
2.2.5.3.5. To enable detection of the maximum number of infected and diseased
animalsin a herd or in a region, Member Statesmay modify the
criteria for the interpretation of the test in order to achieve improved
test sensitivity considering all inconclusive reactions referred in
2.2.5.3.1(b) and 2.2.5.3.2(b) as positive reactions.
3. SUPPLEMENTARY TESTING
To enable detection of the maximum number of infected and diseased
animalsin a herd or in a region, Member Statesmay authoris e the
employ of the gamma-interferon assay referred in the OIE Manual of
Standards for Diagnostic Tests and Vaccines, 4th Edition, 2000,
Chapter 2.3.3. (bovine tuberculosis), in addition to the tuberculin test.
4. STATE INSTITUTES AND NATIONAL REFERENCE LABORA-
TORIES
4.1.Tasksandresponsibilities
The State Institutes and Reference Laboratories included in paragraph
3.2 shall be responsible for the official testing of tuberculins or
reagentsincluded in paragraph 2 and 3 in their res pective Statesto
ensure each of these tuberculins or reagents are adequate in relation
to the standards above referred.
4.2.ListofStateinstitutesandnationalreference laboratories
1. Germany:
Paul-Ehrlich Institut (PEI), Bundesamt für Sera und Impstoffe, D-
23207 Langen; Bundesinstitut für gesundheitlichen Verbrau-
cherschutz und Veterinärmedizin — Bereich Jena — D-07743
Jena;
2. Belgium:1964L0432 — EN — 01.05.2004 — 009.001 — 31
▼M49
Institut Scientifique de la Santé Publique — Louis Pasteur, 14
Rue Juliette Wytsman — B 1050 Bruxelles — Belgique;
3. France:
Laboratoire national des médicaments vétérinaires, Fougères;
4. Grand Duchy of Luxembourg:
Institute of the supplying country;
5. Italy:
Istituto superiore di sanità, Rome;
6. Netherlands:
Centraal Instituut voor Dierziekte Controle Lelystad (CIDC-
Lelystad), Lelystad;
7. Denmark:
Danmarks Veterinærinstitut, Bülowsvej 27, DK-1790 København;
8. Ireland:
Institute of the supplying country;
9. United Kingdom:
Veterinary Laboratory Agency, Addlestone, Weybridge;
10. Greece:
Κέντρο Κτηνιατρικών Ιδρυµάτων, Νεαπόλεως 25, 153 10
Αθήνα;
11. Spain:
Laboratorio de Sanidad y producción animal de Granada;
12. Portugal:
Laboratorio Nacional de Investigação Veterinária, Lisbon;
13. Austria:
Bundesanstalt für veterinärmedizinische Untersuchungen,
Mödling;
14. Finland:
Eläinlääkintä — ja elintarviketutkimus — laitos — Forskningsan-
stalten för veterinärmedicin och livsmedel, Helsinki;
15. Sweden:
Statens veterinärmedicinska anstalt, Uppsala;
16. Czech Republic: Státní veterinární ústav Praha, Sídlištní 24/136,
165 03 Praha 6; Ústav pro státní kontrolu veterinárních biopre-
parátůal éčiv, Hudcova 56 A, 621 00 Brno;
17. Estonia: Ravimiamet, Ravila 19, 504 11 Tartu;
18. Cyprus:ΕργαστήριοΑναφοράςγιατιςΑσθένειεςτωνΖώων ,
ΚτηνιατρικέςΥπηρεσίες 1417Λευκωσία ,(National Reference
Laboratory for Animal Health Veterinary Services, CY-1417
Nicosia);
19. Latvia: Valsts veterin ārmedic īnas diagnostikas centrs, Lejupes
iela 3, LV-1076 R īga;
20. Lithuania: Nacionalin ėveterinarijoslaboratorija, J. Kairi ūkščio g.
10, LT-2021 Vilnius;
21. Hungary: Állatgyógyászati Oltóanyag-, Gyógyszer- és Takarmá-
nyellen őrzőIntézet (ÁOGYTI), Pf. 318., Szállásu. 7., H-1475
Budapest;
22. Malta:
Institute of the supplying country;
23. Poland: Laboratorium Zakładu Mikrobiologii Pa ństwowego Insty-
tutu Weterynaryjnego, Al. Partyzantów 57, PL-24-100 Puławy;
24. Slovenia: Nacionalni veterinarski inštitut, Gerbi čeva 60, SI-1000
Ljubljana;
25. Slovakia: Ústav štátnej kontroly veterinárnych biopreparátov a
liečiv, Biovetská 34, SK-949 01 Nitra.1964L0432 — EN — 01.05.2004 — 009.001 — 32
▼A4
▼M48
ANNEX C
BRUCELLOSIS
1. IDENTIFICATION OF THE AGENT
The demonstration by modified acid-fast or immunospecific staining of
organisms of Brucella morphology in abortion material, vaginal
discharges or milk provides presumptive evidence of brucellosis, espe-
cially if supported by serological tests.
After isolation, the species and biovar should be identified by phage
lysis and/or oxidative metabolism tests, cultural, biochemical and sero-
logical criteria.
The techniques and media used, their standardisation and the interpreta-
tion of results must conform to that specified in the OIE Manual of
Standards for Diagnostic Tests and Vaccines, Fourth Edition, 2000,
Chapter 2.3.1 (bovine brucellosis), Chapter 2.4.2 (caprine and ovine
brucellosis) and Chapter 2.6.2 (porcine brucellosis).
2. IMMUNOLOGICAL TESTS2.1.Standards
2.1.1. The Brucella abortus biovar 1 Weybridge strain No 99 or USDA strain
1119-3 must be used for the preparation of all antigens used in the rose
bengal test (RBT), serum agglutination test (SAT), complement fixation
test (CFT) and the milk ring test (MRT).
2.1.2. The standard reference serum for the RBT, SAT, CFT and MRT is the
OIE international reference standard serum (OIEISS) formerly named
WHO second international anti- Brucella abortus Serum (ISAbS).
2.1.3. The standard reference sera for ELISAs are:
— the OIEISS,
— the weak positive OIE ELISA standard serum (OIEELISA
WPSS),
— the strong positive OIE ELISA standard serum (OIEELISASPSS),
— The negative OIE ELISA standard serum (OIEELISANSS).
2.1.4. The above listed standard sera are available from the Veterinary
LaboratoriesAgency (VLA), Weybridge, United Kingdom.
2.1.5. The OIEISS, the OIEELISAWPSS, the OIEELISASPSS and the OIEELI-
SANSS are international primary standards from which secondary
reference national standards (‘working standards’) must be established
for each test in each Member State.
2.2.Enzyme-linked immunosorbent assays (ELISAs) or other binding
assaysforthedetectionofbovinebrucellosisinserumormilk
2.2.1. Material and reagents
The technique used and the interpretation of results must have been
validated in accordance with the principleslaid down in Chapter 1.1.3
of the OIE Manual of Standards for Diagnostic Tests and Vaccines,
Fourth Edition, 2000, and should at least include laboratory and diag-
nostic studies.
2.2.2. Standardisation of the test
2.2.2.1. Standardisation of the test procedure for individual serum samples:
(a) a 1/150 pre-dilution (1) of the OIEISS or a 1/2 pre-dilution of the
OIEELISAWPSS or a 1/16 pre-dilution of the OIEELISASPSS made
up in a negative serum (or in a negative pool of sera) should give a
positive reaction;
(b) a 1/600 pre-dilution of the OIEISS or a 1/8 pre-dilution of the
OIEELISAWPSS or a 1/64 pre-dilution of the OIEELISASPSS made
up in a negative serum (or in a negative pool of sera) should give a
negative reaction;
(c) the OIEELISANSS should always give a negative reaction.1964L0432 — EN — 01.05.2004 — 009.001 — 33
(1) For the purpose of this Annex, dilutions given for making up liquid reagents are
expressed as, for example, 1/150 shall mean a 1 in 150 dilution.
▼M48
2.2.2.2. Standardisation of the test procedure for pooled serum samples:
(a) a 1/150 pre-dilution of the OIEISS or a 1/2 pre-dilution of the
OIEELISAWPSS or a 1/16 pre-dilution of the OIEELISASPSS made
up in a negative serum (or in a negative pool of sera) and again
diluted in negative sera by the number of samples making up the
pool should give a positive reaction;
(b) the OIEELISANSS should always give a negative reaction;
(c) the test must be adequate to detect evidence of infection in a single
animal of the group of animals, of which samples of serum have
been pooled.
2.2.2.3. Standardisation of the test procedure for pooled milk or whey samples:
(a) a 1/1000 pre-dilution of the OIEISS or a 1/16 pre-dilution of the
OIEELISAWPSS or a 1/125 pre-dilution of the OIEELISASPSS
made up in a negative serum (or in a negative pool of sera) and
again diluted 1/10 in negative milk should give a positive reaction;
(b) the OIEELISANSS diluted 1/10 in negative milk should always give
a negative reaction;
(c) the test must be adequate to detect evidence of infection in a single
animal of the group of animals, of which samples of milk or whey
have been pooled.
2.2.3. Conditions for use of the ELISAs for diagnosis of bovine brucellosis:
2.2.3.1. Using the abovementioned calibrating conditions for ELISAs on serum
samples, the diagnostic sensitivity of ELISA shall be equal or greater
than the RBT or CFT taking into account the epidemiological situation
under which it isemployed.
2.2.3.2. Using the abovementioned calibrating conditions for ELISA on pooled
milk samples, the diagnostic sensitivity of ELISA shall be equal or
greater than the MRT taking into account not only the epidemiological
situation but also the average and expected extreme husbandry systems.
2.2.3.3. Where ELISAs are used for certification purposes in accordance with
Article 6(1) or for the establishment and maintenance of a herd status
in accordance with Annex A(II)(10), pooling of samples of serum
must be carried out in such a way that the test results can be undoubt-
edly related to the individual animal included in the pool. Any
confirmatory test must be carried out on samples of serum taken from
individual animals.
2.2.3.4. The ELISAs may be used on a sample of milk 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 for examination can be undoubtedly related to the individual
animals from which the milk derived. Any confirmatory test must be
carried out on samples of serum taken from individual animals.
2.3.Complementfixationtest(CFT)
2.3.1. The antigen represents a bacterial suspension in phenol-saline (NaCl
0,85 % (m/v) and phenol at 0,5 % (v/v)) or in a veronal buffer. Anti-
gensmay be delivered in the concentrated s tate provided the dilution
factor to be used is indicated on the bottle label. The antigen must be
stored at 4 ºC and not frozen.
2.3.2. Serumsmus t be inactivated asfollows :
— bovine serum: 56 to 60 ºC for 30 to 50 minutes,
— porcine serum: 60 ºC for 30 to 50 minutes.
2.3.3. In order to carry out the genuine reaction within the test procedure, a
complement dose higher than the minimum necessary for total haemo-
lysis should be used.
2.3.4. In carrying out the complement fixation test, the following controls
must be made each time:
(a) control of the anti-complementary effect of the serum;
(b) control of the antigen;
(c) control of sensitised red blood cells;
(d) control of the complement;
(e) control using a positive serum of sensitivity at the start of the reac-
tion;
(f) control of the specificity of the reaction using a negative serum.1964L0432 — EN — 01.05.2004 — 009.001 — 34
▼M48
2.3.5. Calculation of results
The OIEISS contains1 000 international CFT units(ICFTU) per ml. If
the OIEISS is tested in a given method the result is given as a titre
(TOIEISS). The test result for the test serum given as titre (TTESTSERUM)
must be expressed in ICFTU per ml. In order to convert the expression
of a titre into ICFTU, the factor (F) necessary to convert a titre of an
unknown test serum (TTESTSERUM) tested by that method into the ICFTU
expression can be found from the formula:
and the content of international CFT units per ml of test serum (ICFTU-
TESTSERUM) from the formula:
2.3.6. Interpretation of results
A serum containing 20 or more ICFTU per ml is considered to be posi-
tive.
2.4.Milkringtest(MRT)
2.4.1. The antigen represents a bacterial suspension in phenol-saline (NaCl
0,85 % (m/v) and phenol at 0,5 % (v/v)) stained with haematoxylin.
The antigen must be stored at 4 ºC and not frozen.
2.4.2. The antigen sensitivity must be standardised in relation to the OIEISS
in such a way that the antigen produces a positive reaction with a 1/500dilution of the OIEISS in negative milk, while a 1/1 000 dilution should
be negative.
2.4.3. The ring test must be made on samples representing the contents of
each milk churn or the content of each bulk tank from the farm.
2.4.4. The milk samples must not have been frozen, heated or subjected to
violent shaking.
2.4.5. The reaction must be carried out using one of the following methods:
— on a column of milk of at least 25 mm height and on a volume of
milk of 1 ml to which either 0,03 ml or 0,05 ml of one of the stan-
dardised stained antigens has been added,
— on a column of milk of at least 25 mm height and on a volume of
milk of 2 ml to which 0,05 ml of one of the standardised stained
antigenshasbeen added,
— on a volume of milk of 8 ml to which 0,08 ml of one of the stan-
dardised stained antigens has been added.
2.4.6 The mixture of milk and antigensmus t be incubated at 37 ºC for 60
minutes, together with positive and negative working standards. A
subsequent 16 to 24 hour incubation at 4 ºC increases the sensitivity
of the test.
2.4.7. Interpretation of results:
(a) negative reaction: coloured milk, colourless cream;
(b) positive reaction:
— identically coloured milk and cream, or
— colourless milk and coloured cream.
2.5.RosebengalplateTest(RBT)
2.5.1. The antigen represents a bacterial suspension in buffered Brucella
antigen diluent at a pH of 3,65 ± 0,05, stained by the use of rose bengal
dye. The antigen shall be delivered ready for use and must be stored at
4 ºC and not frozen.
2.5.2. The antigen shall be prepared without reference to the cell concentra-
tion, but its sensitivity must be standardised in relation to the OIEISS
in such a way that the antigen produces a positive reaction with a serum
dilution of 1/45 and a negative reaction with a dilution of 1/55.
2.5.3. The RBT shall be carried out in the following manner:
(a) serum (20-30 µl) ismixed with an equal volume of antigen on a
white tile or enamel plate to produce a zone approximately 2 cm
in diameter. The mixture isrocked gently for 4 minutesat ambient
temperature, and then observed in a good light for agglutination;1964L0432 — EN — 01.05.2004 — 009.001 — 35
▼M48
(b) an automated method may be used but must be at least as sensitive
and accurate asthe manual method.
2.5.4. Interpretation of results
Any visible reaction is considered to be positive, unless there has been
excessive drying round the edges.
Positive and negative working standards should be included in each
series of tests.
2.6.Serumagglutinationtest(SAT)
2.6.1. The antigen represents a bacterial suspension in phenol-saline (NaCl
0,85 % (m/v) and phenol at 0,5 % (v/v)). Formaldehyde must not be
used.
Antigensmay be delivered in the concentrated s tate provided the dilu-
tion factor to be used is indicated on the bottle label.
EDTA may be added to the antigen suspension to 5 mM final test dilu-
tion to reduce the level of false positives to the serum agglutination test.
Subsequently the pH of 7,2 must be readjusted in the antigen suspen-
sion.
2.6.2. The OIEISS contains1 000 international unitsof agglutination.2.6.3. The antigen shall be prepared without reference to the cell concentra-
tion, but its sensitivity must be standardised in relation to the OIEISS
in such a way that the antigen produces either a 50 % agglutination
with a final serum dilution of 1/600 to 1/1 000 or a 75 % agglutination
with a final serum dilution of 1/500 to 1/750.
It may also be advisable to compare the reactivity of new and
previously standardised batches of antigen using a panel of defined
sera.
2.6.4. The test is performed either in tubes or in microplates. The mixture of
antigen and serum dilutions should be incubated for 16 to 24 hours at
37 ºC.
At least three dilutions must be prepared for each serum. Dilutions of
suspect serum must be made in such a way that the reading of the reac-
tion at the positivity limit is made in the median tube (or well for the
microplate method).
2.6.5. Interpretation of results:
The degree of Brucella agglutination in a serum must be expressed in
IU per ml.
A serum containing 30 or more IU per ml is considered to be positive.
3. COMPLEMENTORY TESTS3.1.Brucellosisskintest(BST)
3.1.1. Conditions for the use of BST
(a) The brucellosis skin test shall not be used for the purpose of certi-
fication for intra-Community trade.
(b) The brucellosis skin test is one of the most specific tests for the
detection of brucellosis in unvaccinated animals, however diagnosis
should not be made on the basis of positive intradermal reactions
alone.
(c) Bovine animals, tested with negative result in one of the serological
tests defined in this Annex and reacting positively to the BST shall
be regarded asinfected.
(d) Bovine animals, tested with positive result in one of the serological
tests defined in this Annex may be subject to a BST in order to
support the interpretation of the serological test results, in particular
where in brucellosis free or officially free herds a cross-reactionwith antibodiesagains t other bacteria cannot be excluded.
3.1.2. The test must be carried out by use of a standardised and defined
brucellosis allergen preparation that does not contain smooth lipopoly-
saccharide (LPS) antigen, as this may provoke non-specific
inflammatory reactions or interfere with subsequent serological tests.
One of such preparation is Brucellin INRA prepared from a non smooth
strain of B. melitensis . The requirementsfor itsproduction are detailed1964L0432 — EN — 01.05.2004 — 009.001 — 36
▼M48
in Section B2 of Chapter 2.4.2. of the OIE Manual of Standardsfor
Diagnostic Tests and Vaccines, Fourth Edition, 2000.
3.1.3. Test procedure
3.1.3.1. A volume of 0,1 ml of brucellosis allergen is injected intradermally into
the caudal fold, the skin of the flank, or the side of the neck.
3.1.3.2. The test is read after 48-72 hours.
3.1.3.3. The skin thickness at the injection site is measured with vernier calli-
persbefore injection and at re-examination.
3.1.3.4. Interpretation of results:
Strong reactions are easily recognised by local swelling and induration.
Skin thickening of 1,5 to 2 mm shall be considered as positive reaction
to the BST.
3.2.Competitiveenzyme-linkedimmunosorbentassay(cELISA)
3.2.1. Conditions for the use of cELISA
(a) The cELISA shall not be used for the purpose of certification for
intra-Community trade.
(b) The cELISA has shown to have a higher specificity than for
example the indirect ELISA and may therefore be used in order to
support the interpretation of the serological test results.
3.2.2. Test procedure
The test shall be carried out in accordance with the prescription in the
OIE Manual of Standards for Diagnostic Tests and Vaccines, Fourth
Edition, 2000, Chapter 2.3.1(2)(a).
4. NATIONAL REFERENCE LABORATORIES
4.1.Tasksandresponsibilities
National reference laboratories shall be responsible for:
(a) the approval of the results of the validation studies demonstrating
the reliability of the test method used in the Member State;
(b) determination of the maximum number of samples to be pooled in
ELISA kitsus ed;
(c) calibration of the standard secondary reference national standard
sera (‘working standards’) against the primary international stan-
dard serum referred to in paragraph 2.1;
(d) quality checksof all antigensand ELISA kitsbatchesus ed in the
Member State;
(e) cooperation within the European Union Network of National Refer-
ence Laboratories for Brucellosis.
4.2.Listofnationalreference laboratories
BELGIUMCentre d'étudeset de recherchesvétérinaireset agrochimiques
(CERVA/CODA)
Groeselenberg 99B-1180 Bruxelles/Brussel
CZECH REPUBLIC
Státní veterinární ústav Olomouc, Jakoubka ze St říbra 1, 779 00
Olomouc
DENMARK
Danish Veterinary Institute
Bulowsvej 27DK-1790 Copenhagen
GERMANY
Bundesinstitut für Gesundheitlichen Verbraucherschutz und Veterinär-
medizin (BGVV)Nationales Veterinärmedizinisches Referenzlabor für Brucellose1964L0432 — EN — 01.05.2004 — 009.001 — 37
▼A4
▼M48
▼M48
Postfach 33 00 13
D-14191 Berlin
ESTONIA
Institute of the supplying country
GREECE
Veterinary Laboratory of Larissa
Department of Microbiology
6th km of National Road Larissa-TrikalaGR-4111 10 Larissa
SPAIN
Laboratorio Central de Veterinaria de Santa Fe
Camino del Jau S/N
E-18320 Santa Fe (Granada)
FRANCE
Laboratoire national et OIE/FAO de référence pour la brucellose
Agence française de sécurité sanitaire des aliments (AFSSA)
BP 67
F-94703 Maisons-Alfort Cedex
IRELAND
Brucellosis Laboratory
Model Farm RoadCork
Ireland
ITALY
Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise
Via Campo Boario
I-64100 Teramo
CYPRUS
Εργαστήριο Αναφοράς για τις Ασθένειες των Ζώων ,
Κτηνιατρικές Υπηρεσίες , 1417Λευκωσία National Reference
Laboratory for Animal Health Veterinary Services
CY-1417 Nicosia
LATVIA
Valsts veterin ārmedic īnas diagnostikas centrs, Lejupes iela 3,
LV-1076 R īga
LITHUANIANacionalin ėveterinarijoslaboratorija, J. Kairi ūkščio g. 10,
LT-2021 Vilnius
LUXEMBOURG
State Laboratory for Veterinarian Medicine
54, avenue Gaston DiderichB.P. 2081
L-1020 Luxembourg
HUNGARY
Állatgyógyászati Oltóanyag-, Gyógyszer- és Takarmányellen őrző
Intézet (ÁOGYTI),
Pf. 318., Szállásu. 7., H-1475 Budapes t
MALTA
Institute of the supplying country
NETHERLANDS
Centraal Instituut voor DierziekteControle
CIDC-Lelystad
Houtribweg 39P.O. Box 20041964L0432 — EN — 01.05.2004 — 009.001 — 38
▼A4
▼M48
▼A4
▼M48
▼A4
▼M48
▼M48
8203 AA Lelystad
Nederland
AUSTRIA
Bundesanstalt für veterinärmedizinische Untersuchungen
Robert-Koch-Gasse 17A-2340 Modling
POLAND
Laboratorium Zakładu Mikrobiologii Pa ństwowego Instytutu Wetery-
naryjnego, Al. Partyzantów 57, PL-24-100 Puławy
PORTUGAL
Laboratório Nacional de Investigação Veterinária (LNIV)
Estrada de Benfica, n.
o701
P-1549-011 Lisboa
SLOVENIA
Nacionalni veterinarski inštitut, Gerbi čeva 60,
SI-1000 Ljubljana
SLOVAKIA
Štátny veterinárny ústav, Pod dráhami 918,
SK-960 86 Zvolen
FINLAND
National Veterinary and Food Research Institute
Hämeentie 57
PO Box 45
FIN-00581 Helsinki
SWEDENNational Veterinary Institute
S-751 89 Uppsala
UNITED KINGDOM
1. FAO/WHO Collaborating Centre for Reference and Research on
Brucellosis
Veterinary LaboratoriesAgency
New Haw
Addlestone
Surrey KT15 3NB
United Kingdom
2. Immunodiagnostics Department
Veterinary SciencesDivis ion
Stoney Road Stormont
Belfast BT4 3SD
United Kingdom1964L0432 — EN — 01.05.2004 — 009.001 — 39
▼A4
▼M48
▼A4
▼M48
▼M42
ANNEX D
CHAPTER I
OFFICIALLY ENZOOTIC-BOVINE-LEUKOSIS-FREE HERDS,
MEMBERSTATESANDREGIONS
A. Officially enzootic-bovine-leukosis-free herd means a herd in which:
(i) there is no evidence, either clinical or as a result of a laboratory test, of
any case of enzootic bovine leukosis in the herd and no such case has
been confirmed in the previoustwo years ; and
(ii) all animalsover 24 monthsof age have reacted negatively during the
preceding 12 monthsto two tes tscarried out in accordance with this
Annex, at an interval of at least four months; or
(iii) it meetsthe requirementsof (i) above and iss ituated in an officially
enzootic-bovine-leukosis-free Member State or region.
B. A herd shall retain officially enzootic-bovine-leukosis-free status provided:
(i) the condition in A(i) continuesto be fulfilled;
(ii) any animalsintroduced into the herd come from an officially enzootic-
bovine-leukosis-free herd;
(iii) all animalsover 24 monthsof age continue to react negatively to a tes t
carried out in accordance with Chapter II at intervalsof three years ;
(iv) breeding animalsintroduced into a herd and originating from a third
country have been imported in accordance with Directive 72/462/EEC.
C. The officially leukosis-free status of a herd is to be suspended if the condi-
tions detailed in B are not fulfilled, or where as a result of laboratory tests or
on clinical grounds one or more bovine animals are suspected of having
enzootic bovine leukosis and the suspect animal(s) are immediately slaugh-
tered.
D. The status is to remain suspended until the following requirements are
complied with:
1. If a single animal in an officially enzootic-bovine-leukosis-free herd has
reacted positively to one of the tests referred to in Chapter II, or where
infection is otherwise suspected in one animal in a herd:
(i) the animal which has reacted positively, and, in the case of a cow,
any calf it may have produced, must have left the herd for slaughter
under the supervision of the veterinary authorities;
(ii) all animalsin the herd more than 12 monthsold have reacted nega-
tively to two serological tests (at least 4 months and less than 12
monthsapart) carried out in accordance with Chapter II three months
at least after removal of the positive animal and any possible
progeny thereof;
(iii) an epidemiological inquiry hasbeen conducted with negative res ults
and the herdslinked epidemiologically to the infected herd have
been subjected to the measures laid down in (ii).
However, the competent authority may grant a derogation from the obli-
gation to slaughter the calf of an infected cow where it was separated
from its mother immediately after calving. In this case, the calf must be
made subject to the requirements provided for in 2(iii).
2. Where more than one animal from an officially enzootic-bovine-leukosis-
free herd has reacted positively to one of the tests referred to in Chapter
II, or where infection has otherwise been suspected in more than one
animal in a herd:
(i) any animals which have reacted positively and, in the case of cows,
their calves, must be removed for slaughter under the supervision of
the veterinary authorities;
(ii) all animalsin the herd aged over 12 monthsmus t react negatively to
two tests carried out in accordance with Chapter II at an interval of
at least four months and no more than 12 months;
(iii) all other animalsin the herd mus t, after identification, remain on the
holding until they are aged over 24 monthsand have been tes ted in
accordance with Chapter II after reaching that age, except that the
competent authority may permit such animals to go directly for
slaughter under official supervision;
(iv) an epidemiological inquiry hasbeen conducted with negative res ults
and any herd linked epidemiologically to the infected herd hasbeen
subjected to the measures laid down in (ii).1964L0432 — EN — 01.05.2004 — 009.001 — 40
▼M44
▼M44
However, the competent authority may grant a derogation from the obli-
gation to slaughter the calf of an infected cow where it was separated
from its mother immediately after calving. In this case, the calf must be
made subject to the requirements provided for in 2(iii).
E. In accordance with the procedure in Article 17 and on the basis of informa-
tion supplied in accordance with Article 8, a Member State or part of a
Member State may be considered officially enzootic-bovine-leukosis-free if:
(a) all the conditionsof paragraph A are fulfilled and at leas t 99,8 % of the
bovine herds are officially enzootic-bovine-leukosis-free;
or
(b) no case of enzootic bovine leukosis has been confirmed in the Member
State or the part of the Member State for the past three years, and the
presence of tumours suspected of being due to EBL is compulsorily noti-
fiable, with investigations of cause being carried out, and
in the case of a Member State, all animals aged over 24 months in at
least 10 % of the herds, selected randomly, have been tested with nega-
tive results in accordance with Chapter II in the previous 24 months, or
in the case of a part of a Member State, all animals aged over 24 months
have undergone a test provided for in Chapter II with negative results in
accordance with Chapter II in the previous24 months ;
or
(c) any other method which demonstrates to a confidence rating of 99 %
that less than 0,2 % of herds were infected.
F. A Member State or a region of a Member State isto retain officially
enzootic-bovine-leukosis-free status if:
(a) all animalss laughtered within the territory of that Member State or
region are submitted to official post-mortem examinations at which all
tumourswhich could be due to the EBL virusare s ent for laboratory
examination,
(b) the Member State reports to the Commission all cases of enzootic
bovine leukosis that occur in the region,
(c) all animals which react positively to any of the tests provided for in
Chapter II are slaughtered and their herds remain subject to restrictions
until re-establishment of their status in accordance with Section D, and
(d) all animalsmore than two yearsold have been tes ted, either once in the
first five years after the status is granted under Chapter II or during the
first five years after the grant of the status under any other procedure
demonstrating with a certainty level of 99 % that less than 0,2 % of
herdshave been infected. However, where no cas e of enzootic bovine
leukosis has been recorded in a Member State or in a region of a
Member State in a proportion of one herd out of 10 000 for at least three
years, a decision may be taken in accordance with the procedure laid
down in Article 17 that routine serological tests may be reduced
provided that all bovine animalsmore than 12 monthsold in at leas t
1 % of herds, selected at random each year, have been subjected to a
test carried out in accordance with Chapter II.
G. The officially enzootic-bovine-leukosis-free status of a Member State or part
of a Member State is to be suspended, in accordance with the procedure inArticle 17 if, as a result of investigations carried out in accordance with
paragraph F above, there is evidence of a significant change in the situation
asregardsenzootic bovine leukos isin a Member State or part of a Member
State which has been recognised as officially enzootic-bovine-leukosis-free.
The officially enzootic-bovine-leukosis-free status may be restored in accor-
dance with the procedure in Article 17 when the criteria laid down by the
same procedure are fulfilled.
CHAPTER II
TESTSFORENZOOTICBOVINELEUKOSIS
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 immu-
nosorbent 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.1964L0432 — EN — 01.05.2004 — 009.001 — 41
▼M42
▼M42
A.Agargelimmune-diffusiontestforenzootic bovineleukosis
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.
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.
(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: StatensVeterinaere 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
10Αθήνα;;
(m) Austria: Bundesanstalt für Tierseuchenbekämpfung, Mödling;
(n) Finland: Eläinlääkintä-ja elintarvikelaitos, Helsinki/Anstalten för
veterinärmedicin och livsmedel, Helsingfors;
(o) Sweden: Statens veterinärmedicinska anstalt, Uppsala;
(p) 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 , 1417Λευκωσία National Reference
Laboratory for Animal Health Veterinary Services,CY-1417
Nicosia;
(s) Latvia: Valsts veterin ārmedic īnasdiagnos tikascentrs
Lejupesiela 3
LV-1076 R īga;
(t) Lithuania: Nacionalin ėveterinarijoslaboratorija,
J. Kairi ūkščio 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, Gerbi čeva 60,
SI-1000 Ljubljana;
(y) Slovakia: Štátny veterinárny ústav, Pod dráhami 918,
SK-960 86 Zvolen.1964L0432 — EN — 01.05.2004 — 009.001 — 42
▼A4
▼M42
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.
4. The reagents for the test shall consist of:
(a) antigen: the antigen must contain specific glycoproteins of enzootic
bovine leukosis virus which has been standardized against the offi-
cial 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
wellsin a circle around it.
Diameter of central well: 4 mmDiameter of peripheral wells: 6 mmDistance 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.
7. Thisres ultsin the following quantitiesbeing obtained:
antigen: 32 µl
control serum: 73 µl
test serum: 73 µl.
8. Incubation must be for 72 hours at room temperature (20 to 27ºC) in a
closed humid chamber.
9. The test may be read at 24 and 48 hours but a final result may not be
obtained before 72 hours:
(a) a test serum is positive if it forms a specific precipitin line with the
BLV antigen and formsa 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 doesnot bend the line of the control
serum;
(c) the reaction cannot be considered conclusive if it:
(i) bendsthe line of the control s erum towardsthe BLV antigen
well without forming a visible precipitin line with the antigen;
or
(ii) if it cannot be read either asnegative or aspos itive.
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.
B.Methodforantigenstandardization
Solutions and materials required
1. 40 ml of 1,6 % agarose in 0,05 M Tris/HCl buffer, pH 7,2 with 8,5 %
NaCl;
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;1964L0432 — EN — 01.05.2004 — 009.001 — 43
▼M42
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;
4. four plastic petri dishes with a diameter of 85 mm;5. a punch with a diameter of 4 to 6 mm;
6. a reference antigen;
7. the antigen which isto be s tandardized;
8. a water bath (56ºC).
Procedure
Dissolve the agarose (1,6 %) in the Tris/HCl buffer by carefully heating to
100ºC. Place in 56ºC water bath for approximately one hour. Also, place the
bovine leukosis serum dilutions in a 56ºC water bath.
Now mix 15 ml of the 56ºC 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
(i) Petri dishes 1 and 3:
well A — undiluted reference antigen,
well B — 1:2 diluted reference antigen,wellsC 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
1. The experiment shall be carried out with two serum dilutions (1:5 and
1:10) in order to achieve optimal precipitation
2. If the precipitation diameter is too small with both dilutions, then the
serum must be further diluted.
3. If the precipitation diameter in both dilutionsistoo large and faint, then a
lower serum must be chosen.
4. The final concentration of the agarose must be 0,8 %; that of the sera 5
and 10 % respectively.
5. Plot the measured diameters in the following coordinate system. The dilu-
tion of the antigen to be tested with the same diameter as the reference
antigen isthe working dilution.
C.Enzyme-linked immunosorbent assay (Elisa) for detecting enzootic
bovineleukosis
1. The material and reagents to be used are as follows:
(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 poly-
clonal 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 anti-
gens. If catching antibodies are coated to the solid phase, theantibodies 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;1964L0432 — EN — 01.05.2004 — 009.001 — 44
▼M42
(h) solutions for the dilution of the test samples for preparations of the
reagentsand for was hing;
(i) a reading system appropriate to the substrate used.
2.Standardization and sensitivity of test
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 insti-
tutes 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
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 animalsfrom which the milk or s era
examined were taken.1964L0432 — EN — 01.05.2004 — 009.001 — 45
▼M42
ANNEX E (I)
(a)Bovinediseases
— Foot-and-mouth disease
— Rabies— Tuberculosis— Brucellosis— Contagiousbovine pleuropneumonia— Enzootic bovine leukosis— Anthrax
(b)Swinediseases
— Rabies
— Brucellosis— Classical swine fever— African swine fever— Foot-and-mouth disease— Swine vesicular disease— Anthrax1964L0432 — EN — 01.05.2004 — 009.001 — 46
▼M42
ANNEX E (II)
— Aujeszky's disease
— Infectiousbovine rhinotracheitis— Brucella suis infection— Transmissible gastro-enteritis1964L0432 — EN — 01.05.2004 — 009.001 — 47
▼M44
ANNEX F
Model 1
1964L0432 — EN — 01.05.2004 — 009.001 — 48
▼M44
►(1)M461964L0432 — EN — 01.05.2004 — 009.001 — 49
▼M44
►(1)M471964L0432 — EN — 01.05.2004 — 009.001 — 50
▼M44
1964L0432 — EN — 01.05.2004 — 009.001 — 51
▼M44
Model 2
1964L0432 — EN — 01.05.2004 — 009.001 — 52
▼M44
►(1)M471964L0432 — EN — 01.05.2004 — 009.001 — 53
▼M44
1964L0432 — EN — 01.05.2004 — 009.001 — 54