1990L0426 — EN — 05.06.2003 — 008.001 — 1
This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents
►B COUNCILDIRECTIVE
of26June1990
onanimalhealthconditionsgoverningthemovementandimportfromthirdcountriesofequidae
(90/426/EEC)
(OJ L 224, 18.8.1990, p. 42)
Amended by:
Official Journal
No page date
►M1Council Directive 90/425/EEC of 26 June 1990 L 224 29 18.8.1990
►M2Council Directive 91/496/EEC of 15 July 1991 L 268 56 24.9.1991
►M3Commission Decision 92/130/EEC of 13 February 1992 L 47 26 22.2.1992
►M4Council Directive 92/36/EEC of 29 April 1992 L 157 28 10.6.1992
►M5Commission Decision 2001/298/EC of 30 March 2001 L 102 63 12.4.2001
►M6Commission Decision 2002/160/EC of 21 February 2002 L 53 37 23.2.2002
►M7Council Regulation (EC) No 806/2003 of 14 April 2003 L 122 1 16.5.2003
Amended by:
►A1Act 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
Corrected by:►
C1Corrigendum, OJ L 296, 27.10.1990, p. 66 (90/426/EEC)
▼B
COUNCILDIRECTIVE
of26June1990
on animal health conditions governing the movement and import
fromthirdcountriesofequidae
(90/426/EEC)
THE COUNCIL OF THE EUROPEAN COMMUNITIES,
Having regard to the Treaty establishing the European Economic
Community, and in particular Article 43 thereof,
Having regard to the proposal from the Commission (1),
Having regard to the opinion of the European Parliament (2),
Having regard to the opinion of the Economic and Social
Committee (3),
Whereas equidae, being live animals, are included in the list of
productsin Annex II to the Treaty;
Whereasin order to ens ure the rational development of equidae
production, thereby increasing productivity in that sector, rulesgoverning the movement of equidae between Member Statesmus t belaid down at Community level;
Whereasthe breeding and rearing of equidae and in particular of hors es
is generally included in the farming sector; whereas it constitutes asource of income for part of the farming population;
Whereasdis paritiesasregardsanimal health conditionsin the Member
Statess hould be eliminated in order to encourage intra-Communitytrade in equidae;
Whereas, in order to encourage the harmonious development of intra-
Community trade, a Community system should be laid down to governimportsfrom third countries ;
Whereasthe conditionsfor the movement on national territory of
equidae bearing an identification document should also be regulated;
Whereas, in order to be the subject of trade, equidae must satisfy
certain animal health requirements, so as to avoid the spreading ofcontagious diseases; whereas it appears in particular appropriate toprovide for a possible regionalization of restrictive measures;
Whereas transport conditions should be laid down for the same reason;Whereas, to ensure that those requirements are satisfied provision must
be made for the issue by an official veterinarian of a health certificateto accompany the equidae to their place of destination;
Whereasthe organization of and the follow-up to the checksto be
carried out by the Member State of destination and the safeguardmeasures to be implemented should be fixed within the framework ofrulesto be laid down for veterinary checksin intra-Community trade inlive animalsin view of the completion of the internal market;
Whereas provision should be made for the possibility of checks by the
Commission; whereas these checks should be carried out in cooperation
with the competent national authorities;
Whereasdefining Community provis ionsapplicable to importsfrom
third countriesrequiresa lis t to be drawn up of third countriesor parts
of third countriesfrom which equidae may be imported;
Whereas the choice of these countries must be based on criteria of a
general nature such as the state of health of the livestock, the organiza-1990L0426 — EN — 05.06.2003 — 008.001 — 2
(1) OJ No C 327, 30. 12. 1989, p. 61.
(2) OJ No C 149, 18. 6. 1990.
(3) OJ No C 62, 12. 3. 1990, p. 46.
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tion and powersof the veterinary s ervicesand the health regulationsin
force;
Whereas, in addition, imports of equidae should not be authorized from
countries infected with contagious or infectious animal diseases whichpresent a risk to Community livestock or which have been free from
such infection for too short a period; whereas such considerations are
also valid for imports from third countries in which vaccination againstsuch diseases is carried out;
Whereasthe general conditionsapplicable to importsfrom third coun-
tries must be supplemented by special conditions drawn up on the basisof the health situation in each of them; whereas the technical natureand the diversity of the criteria on which these special conditionsdepend require for their definition recourse to a flexible and rapid
Community procedure in which the Commission and the Member
Statescooperate clos ely;
Whereas the presentation of a common standard form of certificate
upon import of equidae constitutes an effective means of verifying
that the Community rulesare being applied; whereass uch rulesmayinclude special provisions which may vary according to the thirdcountry concerned, and whereasthismus t be taken into account indrawing up the standard forms of certificates;
Whereas official Community veterinarians should be responsible for
verifying that the requirementsof thisDirective are obs erved, particu-larly in third countries;
Whereasthe checkscarried out upon importation mus t cover the origin
and the state of health of the equidae;
Whereasthe Member Statesmus t be allowed, on the arrival of equidae
in the territory of the Community and during transit to their place ofdestination, to take all measures, including slaughter and disposal,
required for the purpose of safeguarding the health of humans and
animals;
Whereasthe general rulesapplicable to the checksto be carried out on
importation must be defined within an overall context;
Whereasevery Member State mus t have the right to place an
immediate prohibition on importsfrom a third country when s uch
imports may be dangerous for animal health; whereas in such a casecoordination of the attitudesof the Member Stateswith regard to thatthird country must be assured without delay, without prejudice topossible amendments to the list of countries authorized to export to
the Community;
Whereas the provisions of this Directive should be revised in connec-
tion with the completion of the internal market;
Whereas provision should be made for a procedure establishing close
and effective cooperation between the Commission and the Member
State within the Standing Veterinary Committee,
HAS ADOPTED THIS DIRECTIVE:
CHAPTER 1
General provisions
Article 1
ThisDirective laysdown animal health conditionsfor the movement
between Member Statesand import from third countriesof liveequidae.1990L0426 — EN — 05.06.2003 — 008.001 — 3
▼B
Article 2
For the purposes of this Directive:
(a) ‘holding’ means an agricultural or training establishment, a stable
or, generally speaking, any premises or facilities in which equidaeare habitually kept or bred, for whatever use;
(b) ‘equidae’ meanswild or domes ticated animalsof the equine
(including zebras) or asinine species or the offspring of crossings
of those species;
(c) ‘registered equidae’ means any equidae registered as defined in
Directive 90/427/EEC (
1), identified by meansof an identification
document issued by the breeding authority or any other competentauthority of the country where the animal originated whichmanages the studbook or register for that breed of animal or anyinternational association or organization which manages horses forcompetition or racing;
(d) ‘equidae for slaughter’ means equidae intended to be transported
either directly or after transit through a market or an approved
marshalling centre to the slaughterhouse for slaughter;
(e) ‘equidae for breeding and production’ meansequidae other than
those mentioned in (c) and (d);
(f) ‘Member State or third country free from African horse sickness’
meansany Member State or third country in which there hasbeen
no clinical, serological (in unvaccinated equidae) or epidemiolo-gical evidence of African horse sickness on the territoryconcerned in the previoustwo yearsand in which there have beenno vaccinations against the disease during the previous 12 months;
(g) ‘compulsorily notifiable diseases’ means the diseases listed in
Annex A;
(h) ‘official veterinarian’ meansthe veterinarian des ignated by the
competent central authority of a Member State or of a thirdcountry;
(i) ‘temporary admission’ means the status of a registered animal
originating in a third country and admitted into Community terri-
tory for a period of less than 90 days to be fixed by the
Commission in accordance with the procedure laid down in Article24, depending on the health situation in the country of origin.
CHAPTER II
Rulesforthemovementofequidae
Article 3
Member States shall authorize the movement of equidae registered in
their territory or send equidae to another Member State only wherethey satisfy the conditions laid down in Articles 4 and 5.
However, the competent authoritiesin Member Statesof des tination
may grant general or limited exemption in respect of movement of
equidae which:
— are being ridden or taken, for sporting or recreational purposes,
along roadss ituated near internal bordersof the Community,
— are taking part in cultural or similar events or in activities organized
by authorized local bodiess ituated near internal bordersof theCommunity,
— are intended solely for temporary pasturing or work near internal
bordersof the Community.
Member States making use of such authorization shall inform the
Commission of the content of the exemptions granted.1990L0426 — EN — 05.06.2003 — 008.001 — 4
(1) OJ No L 224, 18. 8. 1990, p. 55.
▼B
Article 4
1. Equidae must show no clinical sign of disease at inspection.
Inspection must be carried out in the 48 hours prior to their embarka-
tion or loading. In the case of registered equidae, however, thisinspection shall, without prejudice to Article 6, be required for intra-Community trade only.
2. Without prejudice to the requirementsof paragraph 5 regarding
compulsorily notifiable diseases, the official veterinarian must, at the
time of inspection, be satisfied that there are no grounds — in parti-cular on the basis of declarations by the owner or breeder — forconcluding that the equidae have been in contact with equidae sufferingfrom an infectious or contagious disease during the 15 days immedi-ately preceding inspection.
3. The equidae must not be intended for slaughter under a national
programme of contagious or infectious disease eradication.
4. The equidae must be identified in the following manner:
(i) in the case of registered horses, by means of an identification docu-
ment, asprovided for in Directive 90/427/EEC (
1), which must
certify in particular that Article 5 (5) and (6) have been compliedwith. The offical veterinarian will have to suspend the validity of
thisdocument for the period of the prohibitionsprovided for in
paragraph 5 or in Article 5. The document should, following theslaughter of the registered horse, be returned to the authority whichissued it. The procedure for the implementation of this point shallbe adopted by the Commission in accordance with the procedurelaid down in Article 24;
(ii) for equidae for breeding and production, identification by a method
to be established by the Commission in accordance with the proce-dure laid down in Article 24.
Until such time as this method is in use, the officially approved
national identification methodss hall remain applicable, providedthat they are notified to the Commission and the other MemberStateswithin three monthsof the date on which thisDirective is
adopted.
5. In addition to the requirementslaid down in Article 5, the
equidae must not come from a holding which has been the subject ofone of the following prohibition orders:
(a) if all the animals of species susceptible to the disease located on
the holding have not been slaughtered, the period of prohibitionconcerning the holding of origin must be at least:
— six months in the case of equidae suspected of having
contracted dourine, beginning on the date of the last actual orpossible contact with a sick animal. However, in the case of astallion, the prohibition shall apply until the animal is castrated,
— six months in the case of glanders or equine encephalomyelitis,
beginning on the day on which the equidae suffering from the
disease in question are slaughtered,
— in the case of infectious anaemia, until the date on which, the
infected animalshaving been s laughtered, the remaininganimals have shown a negative reaction to two Coggins testscarried out three monthsapart,
— six months in the case of vesicular stomatitis,— one month from the last recorded case, in the case of rabies,— 15 days from the last recorded case, in the case of anthrax;
(b) if all the animals of species susceptible to the disease located on
the holding have been slaughtered and the premises disinfected,the period of prohibition shall be 30 days, beginning on the dayon which the animals were destroyed and the premises disinfected,1990L0426 — EN — 05.06.2003 — 008.001 — 5
(1) OJ No L 224, 18. 8. 1990, p. 55.
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except in the case of anthrax, where the period of prohibition is 15
days.
The competent authoritiesmay derogate from thes e prohibition
measures for hippodromes and racecourses, and shall notify the
Commission of the nature of any derogations granted.
6. Where a Member State drawsup or hasdrawn up a voluntary or
compulsory control programme for a disease to which equidae aresusceptible, it may present the programme to the Commission, within
six months of notification of this Directive outlining in particular:
— the distribution of the disease on its territory,
— the reasons for the programme, taking into consideration the signif-
icance of the disease and its cost/benefit advantages,
— the geographical area in which the programme will be imple-
mented,
— the status categories to be applied to establishments, the standards
which must be attained for each species and the test procedures to
be used,
— the programme monitoring procedures,— the action to be taken if, for any reason, a holding 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,
— the non-discriminatory nature of trade in the territory of the
Member State concerned with respect to intra-Community trade.
The Commission shall examine the programmes presented by the
Member States. Where appropriate it shall approve them in accordancewith the procedure laid down in Article 24. Any additional guarantees,general or specific, which may be required in intra-Community trade
may be defined in accordance with the same procedure. Such guaran-
tees must not exceed those required by the Member State in its ownterritory.
Programmes submitted by Member States may be amended or supple-
mented in accordance with the procedure laid down in Article 25.
Amendmentsor additionsto programmeswhich have already been
approved or to guaranteeswhich have been defined in accordancewith the second subparagraph may be approved under the same proce-dure.
Article 5
1. A Member State which is not free of African horse sickness
within the meaning of Article 2 (f) may dispatch equidae from thatpart of itsterritory which iscons idered to be infected within themeaning of paragraph 2 of thisArticle only under the conditionss etout in paragraph 3 of thisArticle.
2. (a) A part of the territory of a Member State shall be considered to
be infected with African horse sickness if:
— clinical, serological (in unvaccinated animals) and/or epide-
miological evidence hasrevealed the pres ence of African
horse sickness in the past two years, or
— vaccination against African horse sickness has been carried
out in the past 12 months.
(b) The part of the territory considered to be infected with African
horse sickness must comprise as a minimum:
— a protection zone with a radiusof at leas t 100 km around
any centre of infection,
— a surveillance zone at least 50 km extending beyond the
protection zone, in which no vaccination hasbeen carriedout in the last 12 months.1990L0426 — EN — 05.06.2003 — 008.001 — 6
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▼M4
(c) The rulescontrolling the combat meas uresrelating to the terri-
toriesand zonesreferred to in points(a) and (b) and the relevantderogationsare s pecified in Directive 92/35/EEC (
1).
(d) All vaccinated equidae found in the protection zone must be
registered and identified in accordance with Article 6 (1) of
Directive 92/35/EEC.
The identification document and/or health certificate shall carry
a clear reference to such vaccination.
3. A Member State may dispatch from the territory referred to in
paragraph 2 (b) only equidae which meet the following requirements:
(a) they must be dispatched only during certain periods of the year,
having regard to the activity of vector insects, to be determined in
accordance with the procedure laid down in Article 25;
(b) they must show no clinical symptom of African horse sickness on
the day of the inspection referred to in Article 4 (1);
(c) — if they have not been vaccinated against African horse sickness,
they must have undergone and reacted negatively to a comple-
ment fixation test for African horse sickness as described in
Annex D, on two occasions, with an interval of between 21and 30 days between the two tests, the second of which musthave been carried out during the 10 daysprior to dis patch,
— if they have been vaccinated, they must not have undergone
vaccination during the previoustwo monthsand mus t have
undergone the fixation test described in Annex D at the afore-
mentioned intervalswithout having recorded an increas e in theantibody count. Under the procedure laid down in Article 24,the Commission may, following the opinion of the ScientificVeterinary Committee, recognize other monitoring methods;
(d) they must have been kept in a quarantine station for a minimum
period of 40 daysprior to dis patch;
(e) they must have been protected from vector insects during the
period of quarantine and during transportation from the quarantinestation to the place of dispatch.
Article 6
Member States which implement an alternative control system
providing guaranteesequivalent to thos e laid down in Article 4 (5) as
regardsmovementswithin their territory of equidae and regis teredequidae, in particular by meansof the identification document, maygrant one another derogations from the provisions of the secondsentence of Article 4 (1) and the second indent of Article 8 (1) on areciprocal basis.
They shall notify the Commission thereof.
Article 7
1. The equidae must be transported, as soon as possible, from the
holding of origin either directly or via an approved market or marshal-
ling centre asdefined in Article 3 (6) of Directive 64/432/EEC to the
place of destination in vehicles or containers which have been regularlycleansed and disinfected with a disinfectant at intervals to be fixed bythe Member State of dispatch. The vehicles must be designed in such away that equidae droppings, litter or fodder cannot escape from the
vehicle during transportation. Transportation must be effected in such
a way that the health and well-being of the equidae can be protectedeffectively.
2. The Member State of destination may, on a general or restricted
basis, grant a derogation from some of the requirements of Article 4 (5)1990L0426 — EN — 05.06.2003 — 008.001 — 7
(1) OJ No L 157, 10. 6. 1992, p. 19.▼B
▼B
for any animal bearing a special mark indicating that it is scheduled for
slaughter, provided that the health certificate mentions such derogation.
In the case of granting such a derogation equidae for slaughter must be
transported directly to the designated slaughterhouse and be slaughtered
within five days of arrival at the slaughterhouse.
3. The official veterinarian must record the identification number or
identification document number of the slaughtered animal and forwardto the competent authority of the place of dispatch, at the latter'srequest, an attestation to the effect that the animal has been slaugh-tered.
Article 8
1. Member States shall ensure that:
— registered equidae which leave their holdings are accompanied by
the identification document laid down in Article 4 (4) together —if they are intended for intra-Community trade — with the attesta-tion provided for in Annex B,
— equidae for breeding, production and slaughter are, during their
transportion, accompanied by a health certificate complying with
Annex C to thisDirective.
The certificate, or in the case of an identification document, the form
containing the health particulars, must, without prejudice to Article 6,be drawn up during the 48 hourspreceding their embarkation or els e nolater than the last working day prior to it, in at least one of the officiallanguages of the Member States of dispatch and destination. The dura-tion of validity of the certificate is 10 days. The certificate must consist
of a single sheet.
2. Importsof equidae other than regis tered equidae may be covered
by a single health certificate per consignment rather than by the indivi-dual certificate referred to in the second indent of paragraph 1.
Article 9
The ruleslaid down in Council Directive 90/425/EEC of 26 June 1990
concerning veterinary and zootechnical checksin intra-Communitytrade in certain live animalsand productswith a view to the comple-
tion of the internal market (
1), shall apply in particular to checks at
origin, to the organization of, and follow-up to, the checksto be carriedout by the Member State of destination, and to the safeguard measuresto be implemented.
Article 10
Veterinary experts from the Commission may, to the extent necessary
to ensure uniform application of this Directive and in cooperation with
the competent national authorities, carry out on-the-spot inspections.
The Commission shall inform the Member States of the outcome ofsuch inspections.
The Member States in whose territory an inspection is carried out shall
give the experts all the assistance necessary to carry out their task.
General arrangementsfor the application of thisArticle s hall be
adopted in accordance with the procedure laid down in Article 24.1990L0426 — EN — 05.06.2003 — 008.001 — 8
(1) OJ No L 224, 18. 8. 1990, p. 29.▼M1
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CHAPTER III
Rulesforimportsfromthirdcountries
Article 11
1. Equidae imported into the Community must satisfy the conditions
laid down in Articles12 to 16.
2. Until the date of entry into force of the decisions adopted
pursuant to Articles 12 to 16, the Member States shall apply to imports
of equidae from third countries conditions at least equivalent to thoseresulting from the application of Chapter II.
Article 12
1. In order to be imported, the equidae must come from a third
country or part of a third country appearing on a list included in aseparate column to be added to the list drawn up in accordance withArticle 3 of Directive 72/462/EEC.
2. The proceduresand criteria for the preparation, modification and
publication of the list of third countries or parts of third countries
provided for in Article 3 of Directive 72/462/EEC shall apply to thelist applicable to imports of equidae.
Article 13
1. The equidae must come from third countries:
(a) free from African horse sickness;
(b) which have been free for two yearsfrom Venezuelan equine ence-
phalomyelitis(VEE);
(c) which have been free for six months from dourine and glanders.
2. The Commission may, in accordance with the procedure laid
down in Article 24:
(a) decide that the provisions of paragraph 1 shall apply to only a part
of the territory of a third country.
In the event that the African horse sickness requirements apply on
a regional basis, at the very least the measures laid down in Article5 (2) and (3) must be complied with;
(b) require additional guarantees for diseases alien to the Community.
Article 14
Before the day of loading for transportation to the Member State of
destination, the equidae must have remained without interruption inthe territory or part of the territory of a third country or, in the eventof regionalization, in the part of the territory defined pursuant toArticle 13 (2) (a) for a period to be determined in the decisions to beadopted pursuant to Article 15.
They must come from a holding placed under veterinary supervision.
Article 15
Importation of equidae from the territory of a third country or part
thereof asdefined in accordance with Article 13 (2) (a) on the lis t
drawn up in accordance with Article 12 (1) shall be authorized only ifthe equidae, over and above the requirementsof Article 13:
(a) comply with the animal health requirementsadopted, with refer-
ence to the species in question and the categories of equidae, inaccordance with the procedure laid down in Article 24 for imports
of equidae from that country.
The reference basis for fixing animal health conditions in accor-
dance with paragraph 1 shall be the standards laid down inArticles4 and 5; and1990L0426 — EN — 05.06.2003 — 008.001 — 9
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(b) in the case of a third country not free of vesicular stomatitis or
viral arteritis for at least six months, the equidae must meet thefollowing requirements:
(i) they must come from a holding which has been free of vesi-
cular stomatitis for at least six months and they must havereacted negatively to a serological test prior to dispatch;
(ii) in the case of viral arteritis, male equidae must — notwith-
standing Article 19 (ii) — have reacted negatively to aserological test or to a virus isolation test or to any other testrecognized in accordance with the procedure laid down in
Article 24 which would guarantee freedom from the virus.
In accordance with the procedure laid down in Article 24, and
following the opinion of the Scientific Veterinary Committee,
the Commission may define the categories of male equidae to
which thisrequirement s hall apply.
Article 16
1. The equidae must be identified in accordance with Article 4 (4)
and accompanied by a certificate drawn up by an official veterinarian
of the exporting third country. Thiscertificate mus t:
(a) be issued on the day of loading of the animals for dispatch to the
Member State of destination or, in the case of registered horses, onthe last working day before embarkation;
(b) be drawn up in at least one of the official languages of the Member
States of destination and one of those of the Member State in
which the import inspection is carried out;
(c) accompany the animalsin the original;
(d) attest that the animals satisfy the requirements of this Directive and
those laid down pursuant to this Directive with regard to importa-
tion from third countries;
(e) consist of a single sheet;
(f) be made out for a single consignee or, in the case of animals for
slaughter, for a consignment, provided the animals are properlymarked and identified.
Member States shall inform the Commission if they make use of
thisoption.
2. The certificate must be drawn up on a form complying with a
model established in accordance with the procedure laid down inArticle 24.
Article 17
Checks shall be carried out on the spot by veterinary experts of the
Member States and the Commission to verify whether the provisionsof thisDirective, and in particular thos e of Article 12 (2), are beingapplied in practice.
Should checkscarried out within the termsof thisArticle bring to light
serious facts as against an approved holding, the Commission shall
immediately inform the Member Statesand forthwith adopt a decis ion
provisionally suspending the approval. The final decision shall be takenaccording to the procedure provided for in Article 25.
The experts from the Member States who are to be entrusted with these
checks shall be appointed by the Commission, acting on a proposalfrom the Member States.
These checks shall be made on behalf of the Community, which shall
bear the cost of any expenditure incurred in this connection.
The frequency of and the procedure for these checks shall be deter-
mined in accordance with the procedure laid down in Article 24.1990L0426 — EN — 05.06.2003 — 008.001 — 10
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Article 18
1. Immediately upon arrival in the Member State of destination,
equidae for slaughter shall be taken to a slaughterhouse, either directly
or after transition through a market or a marshalling centre, and, in
accordance with animal health requirements, be slaughtered within aperiod of time specified in the decisions to be adopted pursuant toArticle 15.
2. Without prejudice to any special conditions which may be
adopted in accordance with the procedure laid down in Article 24, the
competent authority of the Member State of destination may, on animal
health grounds, designate the slaughterhouse to which such equidaemust be taken.
Article 19
The Commission, acting in accordance with the procedure laid down in
Article 24:
(i) may decide that importsfrom a third country or part of a third
country are to be confined to particular species or categories;
(ii) shall, notwithstanding Article 15, establish the special conditions
for the temporary entry into Community territory of registered
equidae or equidae intended for special uses or their re-entry intoCommunity territory after being temporarily exported;
(iii) shall determine the conditions for converting temporary entry into
permanent entry.
CHAPTER IV
Finalprovisions
Article 22
The provisions of this Directive, and in particular those contained in
the second sentence of Article 4 (1) and in Articles 6, 8 and 21, shallbe re-examined before 1 January 1993 in the framework of the propo-sals relating to the completion of the internal market, on which the
Council will decide by a qualified majority.
Article 23
The Annexes to this Directive shall be amended by the Commission in
accordance with the procedure provided for in Article 25.
Article 24
1. The Commission shall be assisted by the Standing Committee on
the Food Chain and Animal Health set up pursuant to Article 58 ofRegulation (EC) No 178/2002 (
1).
2. Where reference ismade to thisArticle, Articles5 and 7 of Deci-
sion 1999/468/EC (2) 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 25
1. The Commission shall be assisted by the Standing Committee on
the Food Chain and Animal Health.1990L0426 — EN — 05.06.2003 — 008.001 — 11
(1) OJ L 31, 1.2.2002, p. 1.
(2) OJ L 184, 17.7.1999, p. 23.▼M2
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2. Where reference ismade to thisArticle, Articles5 and 7 of Deci-
sion 1999/468/EC shall apply.
The period laid down in Article 5(6) of Decision 1999/468/EC shall be
set at 15 days.
Article 26
Article 34 of Directive 72/462/EEC shall apply to the requirements set
out in Chapter III of thisDirective.
Article 27
Member States shall bring into force the laws, regulations and admin-
istrative provisions necessary to comply with this Directive no laterthan 1 January 1992. They shall forthwith inform the Commissionthereof.
Article 28
This Directive is addressed to the Member States.1990L0426 — EN — 05.06.2003 — 008.001 — 12
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▼B
ANNEX A
COMPULSORILYNOTIFIABLEDISEASES
The following diseases are compulsorily notifiable:
— Dourine
— Glanders— Equine encephalomyelitis(of all types , including VEE)— Infectiousanaemia— Rabies
— Anthrax
— African horse sickness— Vesicular stomatitis1990L0426 — EN — 05.06.2003 — 008.001 — 13
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ANNEX B
►(1)M51990L0426 — EN — 05.06.2003 — 008.001 — 14
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ANNEX C
1990L0426 — EN — 05.06.2003 — 008.001 — 15
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►(1)A1
►(2)M51990L0426 — EN — 05.06.2003 — 008.001 — 16
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ANNEX D
AFRICANHORSESICKNESS
DIAGNOSIS
Reagents for the enzyme-linked immunosorbent assays (ELISA) described
below may be obtained from the European Community Reference Laboratory
or the OIE Reference Laboratories for African horse sickness.
1. COMPETITIVE ELISA FOR THE DETECTION OF ANTIBODIES TO
AFRICAN HORSE SICKNESS VIRUS (AHSV) (PRESCRIBED TEST)
Competitive ELISA is used to detect specific AHSV antibodies in sera from
any species of equidae. The broad spectrum, polyclonal, immune anti-AHSV
guinea-pig serum (hereinafter ‘guinea-pig antiserum’) is serogroup specific
and is able to detect all known serotypes of AHS virus.
The principle of the test is the interruption of the reaction between AHSV
antigen and a guinea-pig antiserum by a test serum sample. AHSV antibodies
in the test serum sample will compete with those in the guinea-pig antiserum
resulting in a reduction in the expected colour (following the addition of
enzyme labelled anti-guinea-pig antibody and substrate). Sera can be tested
at a single dilution of 1 in 5 (spot test method) or may be titrated (serum
titration method) to give dilution end-points. Inhibition values higher than
50 % may be regarded aspos itive.
The test protocol described hereinafter is used in the Regional Reference
Laboratory for African horse sickness in Pirbright, United Kingdom.
1.1.Testprocedure
1.1.1.Preparation of plates
1.1.1.1. Coat ELISA plateswith AHSV antigen extracted from infected
cell culturesand diluted in carbonate/bicarbonate buffer, pH
9,6. Incubate the ELISA platesovernight at 4 ºC.
1.1.1.2. Wash plates three times by flooding and emptying the wells
with phosphate buffered saline (PBS), pH 7,2 to 7,4 pH, and
blot dry on adsorbent paper.
1.1.2.Control wells
1.1.2.1. Titrate the positive control sera in a twofold dilution series,
from 1 in 5 to 1 in 640, across column 1 in blocking buffer
(PBS containing 0,05 % (v/v) Tween-20, 5,0 % (w/v)
skimmed-milk powder (Cadbury's MarvelTM) and 1 % (v/v)
adult bovine serum) to give a final volume of 50 µl/well.
1.1.2.2. Add 50 µl of the negative control serum at a dilution of 1 in 5
(10µl serum + 40 µl blocking buffer) to wellsA and B of
column 2.
1.1.2.3. Add 100 µl/well of blocking buffer to wellsC and D of column
2 (blank).
1.1.2.4. Add 50 µl of blocking buffer to wellsE, F, G and H of column
2 (guinea pig control).
1.1.3.Spot test method
1.1.3.1. Add a 1 in 5 dilution of each test serum in blocking buffer to
duplicate wellsof columns3 to 12 (10 µl sera + 40 µl blocking
buffer).
or
1.1.4.Serum titration method
1.1.4.1. Prepare a twofold dilution series of each test sample (1 in 5 to 1
in 640) in blocking buffer across eight wells of single columns
(3 to 12).
then
1.1.5. Add 50 µl of guinea pig antisera, pre-diluted in blocking buffer,
to all wellsexcept the blank wellsof the ELISA plate (all wells
now contain a final volume of 100 µl).
1.1.5.1. Incubate for 1 hour at 37 ºC on an orbital shaker.1990L0426 — EN — 05.06.2003 — 008.001 — 17
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1.1.5.2. Wash plates three times and blot dry as before.
1.1.5.3. Add 50 µl of rabbit anti-guinea-pig horseradish peroxidase
(HRP) conjugate pre-diluted in blocking buffer to each well.
1.1.5.4. Incubate for 1 hour at 37 ºC on an orbital shaker.
1.1.5.5. Wash plates three times and blot dry as before.1.1.6.Chromogen
Prepare the chromogen OPD (OPD = ortho-phenyldiamine)
solution according to the manufacturers instructions (0,4 mg/
ml in sterile distilled water) just before use. Add substrate
(hydrogen peroxide = H
2O2) to give a final concentration of
0,05 % (v/v) (1 in 2000 of a 30 % solution of H2O2). Add
50µl of the OPD solution to each well and leave plates on the
bench for 10 minutesat ambient temperature. Stop the reaction
by the addition of 50 µl/well of 1M sulphuric acid (H2SO4).
1.1.7.Reading
Read spectrophotometrically at 492 nm.
1.2.Expressionofresults
1.2.1. Using a software package print out the optical density (OD)
values, and the percentage inhibition (PI) for test and control
sera based on the mean value recorded in the four guinea pig
control wells. The data expressed as OD and PI values are used
to determine whether the test has performed within acceptable
limits. The upper control limits (UCL) and lower control limits
(LCL) for the guinea pig control are between OD values1,4 and
0,4 respectively. The end-point titre for the positive control based
on 50 % PI should be 1 in 240 (within a range from 1 in 120 to 1
in 480). Any plate that failsto conform to the above criteria mus t
be rejected. However, if the positive control serum titre is greater
than 1 in 480 and the test samples are still negative then the
negative test samples can be accepted.
The duplicate negative control serum wells and the duplicate
blank wellss hould record PI valuesbetween + 25 % and – 25 %,
and between + 95 % and + 105 %, respectively. Failure to be
within these limits does not invalidate the plate but does suggest
that background colour isdeveloping.
1.2.2. The diagnostic threshold (cut-off value) for test sera is 50 % (PI
50 %). Samplesrecording PI valuesgreater than 50 % are
recorded aspos itive. Samplesrecording PI valueslower than
50 % are recorded asnegative.
Samplesthat record PI valuesabove and below the thres hold for
the duplicate wells are considered doubtful. Such samples may be
re-tested in the spot test and by titration. Positive samples may
also be titrated to provide an indication of the degree of posi-tivity.
Spot test layout
1 2 3456789 1 0 1 1 1 2
+ve
cont.Test sera
A 1:5 –ve cont. 31 32 33 34 35 36 37 38 39 40
B 1:10 –ve cont. 31 32 33 34 35 36 37 38 39 40C 1:20 Blank
D 1:40 Blank
E 1:80 GP cont.
F 1:160 GP cont.
G 1:320 GP cont. 1 2 3 4 5 6 7 8 9 101990L0426 — EN — 05.06.2003 — 008.001 — 18
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1 2 3456789 1 0 1 1 1 2
+ve
cont.Test sera
H 1:640 GP cont. 1 2 3 4 5 6 7 8 9 10
–ve cont = negative control.
+ve cont = positive control.
GP cont = guinea pig control.
Test sera
1 2 3 456789 1 0 1 11 2
+ve
cont.Test sera
A 1:5 –ve cont. 1:5 1:5
B 1:10 –ve cont. 1:10 1:10C 1:20 Blank 1:20 1:20
D 1:40 Blank 1:40 1:40
E 1:80 GP cont. 1:80 1:80
F 1:160 GP cont. 1:160 1:160
G 1:320 GP cont. 1:320 1:320H 1:640 GP cont. 1:640 1:640
–ve cont = negative control.
+ve cont = positive control.
GP cont = guinea pig control.
2. INDIRECT ELISA FOR THE DETECTION OF ANTIBODIES TO
AFRICAN HORSE SICKNESS VIRUS (AHSV) (PRESCRIBED TEST)
The test described hereinafter is in accordance with the test description in
Chapter 2.1.11 of the OIE Manual of Standards for Diagnostic Tests and
Vaccines , fourth edition, 2000.
The recombinant VP7 protein hasbeen us ed asantigen for AHS virusanti-
body determination with a high index of sensitivity and specificity. Other
advantagesare that it iss table and not infective.
2.1.Testprocedure
2.1.1.Solid phase
2.1.1.1. ELISA platesare coated with recombinant AHSV-4 VP7
diluted in carbonate/bicarbonate buffer, pH 9,6. Incubate plates
overnight at 4 ºC.
2.1.1.2. Wash the plates five times with distilled water containing
0,01 % (v/v) Tween 20 (washing solution). Gently tap the
plates onto absorbent material to remove any residual wash.
2.1.1.3. Block the plates with phosphate buffered saline (PBS) + 5 %
(w/v) skimmed milk (Nestlé Dry Skim MilkTM), 200 µl/well,
for 1 hour at 37 ºC.
2.1.1.4. Remove the blocking solution and gently tap the plates onto
absorbent material.
2.1.2.Test samples
2.1.2.1. Serum samples to be tested, and positive and negative control
sera, are diluted 1 in 25 in PBS + 5 % (w/v) skimmed milk1990L0426 — EN — 05.06.2003 — 008.001 — 19
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+ 0,05 % (v/v) Tween 20, 100 µl per well. Incubate for 1 hour
at 37 ºC.
For titration, make a twofold dilution series from 1 in 25
(100 µl/well), one serum per plate column, and do the same
with positive and negative controls. Incubate for 1 hour at 37 ºC.
2.1.2.2. Wash the plates as described in step 2.1.1.2.
2.1.3.Conjugate
2.1.3.1. Dispense 100 µl/well of horseradish-peroxidase (HRP) -conju-
gated anti-horse gamma-globulin diluted in PBS + 5 % milk
+ 0,05 % Tween 20, pH 7,2. Incubate for 1 hour at 37 ºC.
2.1.3.2. Wash the plates as described in step 2.1.1.2.2.1.4.Cromogen/Substrate
2.1.4.1. Add 200 µl/well of chromogen/substrate solution (10 ml of
80,6 mM DMAB (dimethyl aminobenzaldehyde) + 10 ml of
1,56 mM MBTH (3-methyl-2-benzo-thiazoline hydrazone
hydrochlorid) + 5 µlH
2O2)
Colour development iss topped by adding 50 µlo f3 NH2SO4after approximately 5 to 10 minutes(before the negative control
beginsto be coloured).
Other chromogens such as ABTS (2,2'-Azino-bis-[3-ethylben-
zothiazoline-6-sulphonic acid]), TMB (tetramethyl benzidine),
or OPD (ortho-phenyldiamine) can also be used.
2.1.4.2. Read the platesat 600 nm (or 620 nm).
2.2.Interpretationoftheresults
2.2.1. Calculate the cut-off value by adding 0,6 to the value of the nega-
tive control (0,6 isthe s tandard deviation derived with a group of
30 negative sera).
2.2.2. Test samples giving absorbance values lower than the cut-off are
regarded asnegative.
2.2.3. Test samples giving absorbance values greater than the cut-off
+ 0,15 are regarded aspos itive.
2.2.4. Test samples giving intermediate absorbance values are doubtful
and a second technique must be employed to confirm the result.
3. BLOCKING ELISA FOR THE DETECTION OF ANTIBODIES TO
AFRICAN HORSE SICKNESS VIRUS (AHSV) (PRESCRIBED TEST)
The blocking ELISA is designed to detect specific AHSV antibodies in sera
from any susceptible species. VP7 is the major, antigenic, viral protein of
AHSV, and is conserved within the nine serotypes. Because the monoclonal
antibody (Mab) is also directed against the VP7, the assay will give a high
level of sensitivity and specificity. Further, the recombinant VP7 antigen is
completely innocuousand therefore guaranteesa high degree of s afety.
The principal of the test is the interruption of the reaction between the
recombinant VP7, asthe antigen bound to the ELISA plate and the conju-
gated Mab specific for the VP7. Antibody in the test sera will block the
reaction between the antigen and the Mab resulting in a reduction in colour.
The test described hereinafter is carried out in the European Community
Reference Laboratory for African horse sickness in Algete, Spain.
3.1.Testprocedure
3.1.1.ELISA plates
3.1.1.1. Coat ELISA plateswith recombinant AHSV-4 VP7 diluted in
carbonate/bicarbonate buffer, pH 9,6. Incubate overnight at 4 ºC.
3.1.1.2. Wash the plates five times with phosphate buffered saline
(PBS) containing 0,05 % (v/v) Tween 20 (PBST).
3.1.1.3. Stabilise the plate by treatment with a stabilising solution (in
order to allow long term storage at 4 ºC without loss of activity)
and blot dry onto adsorbent material.1990L0426 — EN — 05.06.2003 — 008.001 — 20
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3.1.2.Test samples and controls
3.1.2.1. For screening: dilute test sera and controls 1 in 10 directly on
the plate in PBST to give a final volume 100 µl/
well. Incubate for 1 hour at 37 ºC.
3.1.2.2. For titration: prepare a twofold dilution series of test sera
and positive controls (100 µl/well) from 1 in
10 to 1 in 1 280 across eight wells. Negative
control istes ted at 1 in 10 dilution.
3.1.3.Conjugate
Add 50 µl/well of pre-diluted horseradish-peroxidase (HRP)
-conjugated Mab (monoclonal antibodiess pecific for VP7) to
each well and mix gently to ensure homogeneity. Incubate for
30 minutesat 37 ºC.
3.1.4. Wash the plates five times with PBST and blot dry as above.3.1.5.Chromogen/Substrate
Add 100 µl/well of chromogen/substrate solution (1 ml of
ABTS (2,2'-Azino-bis-[3-ethylbenzothiazoline-6-sulphonic
acid]) 5 mg/ml + 9 ml of substrate buffer (0,1 M Phosphate-
Citrate buffer of pH 4 containing 0,03 % H
2O2] and incubate
for 10 minutesat room temperature. Colour development is
stopped by adding 100 µl/well of 2 % (w/v) SDS (sodium
dodecyl sulphate).
3.1.6.Reading
Read at 405 nm in an ELISA reader.
3.2.Interpretationoftheresults
3.2.1.Assay validation
The test is valid when the optical density (OD) of negative
control (NC) ishigher than 1,0 and the OD of pos itive control
(PC) islower than 0,2.
3.2.2.Cut-off calculation
Positive cut-off = NC - ((NC - PC) x 0,3)
Negative cut-off = NC - ((NC - PC) x 0,2)
Where, NC isthe OD of the negative control and PC the OD of
positive control.
3.2.3.Interpretation of results
Samples with OD lower than positive cut-off should be consid-
ered as positives to AHSV antibodies.
Samples with OD higher than negative cut-off should be consid-
ered negativesfor AHSV antibodies .
Samples with OD between these two values should be considered
doubtful and the animals re-sampled after two to three weeks.1990L0426 — EN — 05.06.2003 — 008.001 — 21