EUR-Lex - 01979D0542-20050101 - EN

COUNCIL DECISION

of 21 December 1976

(79/542/EEC)

▼B

THE COUNCIL OF THE EUROPEAN COMMUNITIES,

Having regard to the Treaty establishing the European Economic Community,

Having regard to Council Directive 72/462/EEC of 12 December 1972 on health and veterinary inspection problems upon importation of bovine animals and swine and fresh meat from third countries ( 1 ), as last amended by Directive 77/98/EEC ( 2 ), and in particular Article 3 (1) thereof,

Having regard to the proposal from the Commission,

Whereas the system laid down in Directive 72/462/EEC is based on the establishment of a list of the third countries or parts of third countries from which the Member States authorize imports of bovine animals and swine and of fresh meat of bovine animals, swine, sheep, goats and domestic solipeds, or of one or more of these categories of animals or categories of fresh meat;

Whereas in order to decide in respect both of the animals and of fresh meat whether a country or part of a country may be included in the list, particular account is taken of the criteria set out in Article 3 (2) of the abovementioned Directive;

Whereas the countries listed in the Annex to this Decision which traditionally supply the Member States may be considered to satisfy these criteria;

Whereas, however, this list is drawn up subject to such amendments or additions as may be made to it in accordance with the procedure laid down in Article 30 of Directive 72/462/EEC; whereas it may prove necessary in the light of further information to limit or extend the authorizations for importing certain categories of animals and fresh meat; whereas, it may also be necessary in certain cases in respect both of the animals and of fresh meat to specify the parts of countries from which imports will be authorized;

Whereas, although the list of third countries forms one of the bases of the Community arrangements applicable to imports from third countries laid down in Directive 72/462/EEC other measures, particularly concerning hygiene and veterinary inspection, will have to be taken in order to define these arrangements; whereas, consequently, it is important to facilitate the coordinated implementation of all these measures,

HAS ADOPTED THIS DECISION:

Article 1

Subject matter and scope

This Decision establishes the sanitary conditions for the importation into the Community of live animals excluding equidae, and for the importation of fresh meat and meat products of such animals, including equidae, but excluding meat preparations.

This Decision shall not apply to imports of non-domesticated animals for shows or exhibitions where such animals are not regularly kept or bred, and those non-domesticated animals forming part of circuses, or intended for scientific including conservation or experimental purposes in a body, institute or centre that has been approved in accordance with Annex C to Directive 92/65/EEC.

Imports of animals and fresh meat authorised in accordance with this Decision shall remain subject to other provisions that have been adopted, or may be adopted, under European food law.

Article 2

Definitions

For the purposes of this Decision, the following definitions shall apply:

(a) ‘animals’: means land mammals of the species belonging to the taxa Proboscidea and Artiodactyla, and their crossbreeds;

(b) ‘holding’: means a farm or other officially supervised agricultural, industrial or commercial undertaking, including zoos, amusement parks and wildlife or hunting reserves where animals are regularly kept or bred;

(c) ‘trimmed offal’: means offal from which the bones, the cartilage, the trachea and main bronchi, the lymphatic glands and adhering connective tissue, the fat and the mucus have been completely removed; in the case of meat from domestic bovine animals, the whole masseter muscles, incised in accordance with point 41(a) of Chapter VIII of Annex I to Council Directive 64/433/EEC, are also considered as trimmed offal.

Article 3

Conditions for importation of live animals into the Community

Imports into the Community of live animals shall only be allowed if such animals comply with Articles 4, 5 and 6.

Article 4

Place of origin of live animals

The animals shall come from the territory of a third country or a part thereof as listed in columns 1, 2 and 3 of the table set out in Part 1 of Annex I for which, in the corresponding column 4, there is a specific model of veterinary certificate designated for these animals.

Article 5

Specific conditions

The animals shall meet the requirements set out in the appropriate certificate established in accordance with the corresponding model certificate drawn up under Part 2 of Annex I, taking into account the specific conditions indicated in column 6 of the table set out in Part 1 of Annex I, and, if so indicated in column 5 of the table, they shall also meet any supplementary guarantees required in that certificate.

If required by the Member State of destination, the animals concerned shall meet the additional certification requirements mentioned for that Member State and included in the certificate, based on the corresponding model set out in Part 2.

Article 6

Transport of live animals for importation into the Community

1. The animals shall not be loaded in a means of transport carrying other animals that are not destined for the Community or are of a lower health status.

2. During the transport to the Community, the animals shall not be unloaded in the territory of a third country or part of a third country that is not approved for importation into the Community of such animals.

3. During the transport to the Community, the animals shall not be moved by road, railway or on foot through the territory or part of the territory of a third country that is not approved for importation into the Community of such animals.

4. The animals shall arrive at a border inspection post of the Community within 10 days of the date of loading in the exporting third country and be accompanied by a veterinary certificate, drawn up in conformity with the corresponding model, completed and signed by an official veterinarian of the exporting third country.

In the case of transport by sea, the period of 10 days shall be prolonged by the time of the sea journey. For that purpose, a declaration by the master of the ship, drawn up in accordance with the addendum of Part 3A of Annex I, shall be attached in its original form to the veterinary certificate.

Article 7

Conditions to be applied following importation

Following the importation and in accordance with Directive 91/496/EEC,

(i) animals intended for immediate slaughter shall be conveyed without delay to the slaughterhouse of destination where they shall be slaughtered within five working days;

(ii) animals intended for breeding, production or fattening purposes, and animals intended for zoos, amusement parks and hunting or wildlife reserves, shall be conveyed without delay to the holding of destination where they shall remain for a minimum period of 30 days before further movement outside the holding, except in the case of direct dispatch to a slaughterhouse.

Article 8

Conditions for importation of fresh meat into the Community

Imports into the Community of fresh meat intended for human consumption, from the animals as defined in Article 2 and from equidae, shall only be allowed if such meat complies with Articles 9 to 11.

Article 9

Place of origin of fresh meat

The fresh meat shall come from the territory of a third country or a part thereof as listed in columns 1, 2 and 3 of the table set out in Part 1 of Annex II for which, in the corresponding column 4, there is a specific model of veterinary certificate designated for that meat.

Article 10

Specific conditions

The fresh meat shall meet the requirements set out in the appropriate certificate corresponding to the model certificate drawn up under Part 2 of Annex II, taking into account the specific conditions indicated in column 6 of the table set out in Part 1 of Annex II, and, if so indicated in column 5 of the table, it shall also meet the supplementary guarantees requested in that certificate.

Article 11

Presentation of fresh meat at a Community border inspection post

The fresh meat shall be presented at a Community border inspection post accompanied by a veterinary certificate, drawn up in conformity with the corresponding model, completed and signed by an official veterinarian of the exporting third country.

Article 12

Conditions to be applied following importation

1. Following importation, the following categories of fresh meat shall be conveyed without delay to the processing establishment of destination, in accordance with Directive 97/78/EC:

(a) unskinned carcases of wild cloven-hoofed game intended for human consumption after further processing;

(b) trimmed offal of domestic bovine animals intended for human consumption as meat-based products after further heat-treatment by cooking to a core temperature of at least 80 °C, or sterilised in hermetically sealed containers in a way as to achieve a value of Fo ≥ 3.

2. For the categories of products referred to in paragraph 1(b), the establishment of destination shall be an establishment specifically approved and registered for processing those products by the Member State in which the establishment is situated.

3. In accordance with the procedures established by Decision 2001/106/EC, Member States shall communicate to each other and to the Commission:

(a) the names and addresses of the establishments referred to in paragraph 2 and of the local competent authority responsible for the supervision of these establishments, as well as

(b) the categories of products for which these establishments are approved and registered.

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Article 12a

Member States shall ensure that consignments of meat for human consumption, including minced meat, introduced onto the territory of the Community and which are destined for a third country either by transit immediately or after storage in accordance with Articles 12(4) or 13 of Directive 97/78/EC, and not intended for importation into the Community shall comply with the following requirements:

(a) they shall come from the territory of a third country or a part thereof listed in Annex II, part 1, of this Decision for the import of fresh meat of that species;

(b) they shall meet the specific animal health conditions for the species concerned set out in the corresponding model animal health certificate drawn up pursuant to Annex II, part 2;

(c) they shall be accompanied by an animal health certificate established in accordance with the model laid down in Annex III, signed by an official veterinarian of the competent veterinary services of the third country concerned;

(d) they are certified as acceptable for transit or storage (as appropriate) on the common veterinary entry document by the official veterinarian of the border inspection post of introduction.

Article 12b

1. By way of derogation from Article 12a, Member States shall authorise the transit by road or by rail through the Community, between the designated Community border inspection posts listed in Annex IV, of consignments coming from and destined to Russia directly or via another third country provided that the following conditions are met:

(a) the consignment shall be sealed with a serially numbered seal at the border inspection post (BIP) of entry to the Community by the veterinary services of the competent authority;

(b) the documents accompanying the consignment and referred to in Article 7 of Directive 97/78/EC shall be stamped ‘ONLY FOR TRANSIT TO RUSSIA VIA THE EC’ on each page by the official veterinarian of the competent authority responsible for the BIP;

(d) the consignment is certified as acceptable for transit on the common veterinary entry document by the official veterinarian of the border inspection post of introduction.

2. Unloading or storage, as defined in Article 12(4) or Article 13 of Directive 97/78/EC, of such consignments on Community territory shall not be allowed.

3. Regular audits shall be made by the competent authority to ensure that the number of consignments and the quantities of products leaving the Community territory matches the number and quantities entering.

Article 13

Certification

The veterinary certificates required for the importation of live animals and fresh meat into the Community, as provided for in this Decision, shall be drafted in accordance with the notes set out in Part 2 of Annexes I and II. However, this shall not preclude the use of electronic certification or other agreed systems, harmonised at Community level.

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Article ►M54 14 ◄

This Decision is addressed to the Member States.

ANNEX I

LIVE ANIMALS

PART 1

List of third countries or parts thereof (1)

Country	Code of Territory	Description of territory	Veterinary certificate		Specific conditions
Country	Code of Territory	Description of territory	Model(s)	SG	Specific conditions
1	2	3	4	5	6
BG — Bulgaria	BG-0	Whole country	—
BG — Bulgaria	BG-1	The provinces of Varna, Dobrich, Silistra, Choumen, Targovitchte, Razgrad, Rousse, V. Tarnovo, Gabrovo, Pleven, Lovetch, Plovdic, Smolian, Pasardjik, Sofia distric, Sofia city, Pernik, Kustendil, Blagoevgrad, Sliven, Starazagora, Vratza, Montana and Vidin	BOV-X, BOV-Y, RUM, OVI-X, OVI-Y	A
CA — Canada	CA-0	Whole country	POR-X		IVb IX
CA — Canada	CA-1	Whole country except the Okanagan Valley region of British Columbia described as follows: — from a point on the Canada/United States border 120° 15′ longitude, 49° latitude — northerly to a point 119° 35′ longitude, 50° 30′ latitude — north-easterly to a point 119° longitude, 50° 45′ latitude — southerly to a point on the Canada/United States border 118° 15′ longitude, 49° latitude	BOV-X, OVI-X, OVI-Y, RUM (2)	A	IVb IX
CH — Switzerland	CH-0	Whole country	BOV-X, BOV-Y, OVI-X, OVI-Y, RUM
CH — Switzerland	CH-0	Whole country	POR-X, POR-Y, SUI	B
CL — Chile	CL-0	Whole country	OVI-X, RUM
CL — Chile	CL-0	Whole country	POR-X, SUI	B
GL — Greenland	GL-0	Whole country	OVI-X, RUM		V
HR — Croatia	HR-0	Whole country	BOV-X, BOV-Y, RUM, OVI-X, OVI-Y
IS — Iceland	IS-0	Whole country	BOV-X, BOV-Y, RUM, OVI-X, OVI-Y		I
IS — Iceland	IS-0	Whole country	POR-X, POR-Y	B	I
NZ — New Zealand	NZ-0	Whole country	BOV-X, BOV-Y, RUM, POR-X, POR-Y, OVI-X, OVI-Y		I
PM — St Pierre and Miquelon	PM-0	Whole country	BOV-X, BOV-Y, RUM, OVI-X, OVI-Y, CAM
RO — Romania	RO-0	Whole country	BOV-X, BOV-Y, RUM, OVI-X, OVI-Y		V
(1) Without prejudice to specific certification requirements provided for by any relevant Community agreement with third countries. (2) Exclusively for live animals other than animals belonging to the cervidae species.

Specific conditions

(see footnotes in each certificate)

‘I’

territory where the presence of BSE in native cattle has been assessed as highly unlikely, for the purpose of exporting to the European Community animals certified according to the models of certificate BOV-X and BOV-Y

‘II’

territory recognised as having an official tuberculosis-free status for the purposes of exports to the European Community of animals certified according to the model of certificate BOV-X

‘III’

territory recognised as having an official brucellosis-free status for the purposes of exports to the European Community of animals certified according to the model of certificate BOV-X

‘IVa’

territory recognised as having an official enzootic-bovine-leukosis (EBL) free status for the purposes of exports to the European Community of animals certified according to the model of certificate BOV-X

‘IVb’

territory with approved holdings recognised as having an official enzootic-bovine-leukosis (EBL) free status for the purposes of exports to the European Community of animals certified according to the model of certificate BOV-X

‘V’

territory recognised as having an official brucellosis-free status for the purposes of exports to the European Community of animals certified according to the model of certificate OVI-X

‘VI’

geographical constraints

‘VII’

territory recognised as having an official tuberculosis-free status for the purposes of exports to the European Community of animals certified according to the model of certificate RUM

‘VIII’

territory recognised as having an official brucellosis-free status for the purposes of exports to the European Community of animals certified according to the model of certificate RUM

‘IX’

territory recognised as having an official Aujeszky’s disease -free status for the purposes of exports to the European Community of animals certified according to the model of certificate POR-X.

PART 2

Models of veterinary certificates

Models:

‘BOV-X’

Model of veterinary certificate for domestic bovine animals (Bos taurus, Bison bison, Bubalus bubalis and their cross-breeds) intended for breeding and/or production after importation

‘BOV-Y’

Model of veterinary certificate for domestic bovine animals (Bos taurus, Bison bison, Bubalus bubalis and their cross-breeds) intended for immediate slaughter after importation

‘OVI-X’

Model of veterinary certificate for domestic sheep (Ovis aries) and goats (Capra hircus) intended for breeding and/or production after importation

‘OVI-Y’

Model of veterinary certificate for domestic sheep (Ovis aries) and goats (Capra hircus) intended for immediate slaughter after importation

‘POR-X’

Model of veterinary certificate for domestic porcine animals (Sus scrofa) intended for breeding and/or production after importation

‘POR-Y’

Model of veterinary certificate for domestic porcine animals (Sus scrofa) intended for immediate slaughter after importation

‘RUM’

Model of veterinary certificate for non-domestic animals other than suidae

‘SUI’

Model of veterinary certificate for non-domestic suidae.

‘CAM’

Model of specific attestation for animals imported from St Pierre et Miquelon under the conditions provided for in Part 4 of Annex I.

SG (supplementary guarantees):

‘A’

guarantees regarding bluetongue and epizootic-haemorrhagic disease tests on animals certified according to the model of certificate BOV-X (point 10.8a), OVI-X (point 10.6a) and RUM (point 10.7a)

‘B’

guarantees regarding swine-vesicular disease and classical-swine-fever tests on animals certified according to the model of certificate POR-X (point 10.4a) and SUI (point 10.4a)

‘C’

guarantees regarding brucellosis test on animals certified according to the model of certificate POR-X (point 10.4a) and SUI (point 10.4a)

Notes

(a) Veterinary certificates shall be produced by the exporting country, based on the models appearing in Part 2 of Annex I, according to the layout of the model that corresponds to the animals concerned. They shall contain, in the numbered order that appears in the model, the attestations that are required for any third country and, as the case may be, those supplementary guarantees that are required for the exporting third country or part thereof.

If so requested by the EU Member State of destination, for the animals concerned the additional certification requirements shall be also incorporated in the original form of the veterinary certificate.

(b) A separate and unique certificate must be provided for animals that are exported from a single territory appearing in columns 2 and 3 of Part 1 of Annex I which are consigned to the same destination and transported in the same railway wagon, lorry, aircraft or ship.

(c) The original of each certificate shall consist of a single page, both sides, or, where more text is required, it shall be in such a form that all pages needed are part of an integrated whole and indivisible.

(d) It shall be drawn up in at least one of the official languages of the EU Member State in which the inspection at the border post shall be carried out and of the EU Member State of destination. However, these Member States may allow another Community language instead of their own, accompanied, if necessary, by an official translation.

(e) If for reasons of identification of the items of the consignment (schedule in point 8.2 of the model of certificate), additional pages are attached to the certificate, these pages shall also be considered as forming part of the original of the certificate by the application of the signature and stamp of the certifying official veterinarian on each of the pages.

(f) When the certificate, including additional schedules referred to in (e), comprises more than one page, each page shall be numbered — (page number) of (total number of pages) — at the bottom and shall bear the code number of the certificate that has been designated by the competent authority at the top.

(g) The original of the certificate must be completed and signed by an official veterinarian within 24 hours prior to loading of the consignment for exportation to the Community. In doing so, the competent authorities of the exporting country shall ensure that principles of certification equivalent to those laid down in Council Directive 96/93/EC are followed.

The colour of the signature shall be different to that of the printing. The same rule applies to stamps other than those embossed or watermark.

(h) The original of the certificate must accompany the consignment until it reaches the EU border inspection post.

(i) The certificate shall be valid for 10 days from the date of issuing.

In the case of transport by ship the time of validity is prolonged by the time of the trip in the ship. For this purpose, a declaration by the master of the ship, drawn up in accordance with the addendum to Part 3 of Annex I to this Decision, shall be attached in its original form to the veterinary certificate.

(j) Animals shall not be transported together with other animals that either are not destined to the European Community or are of a lower health status.

(k) During their transport to the European Community, the animals shall not be unloaded in the territory of a country or part of a country that is not approved for imports into the Community of these animals.

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►(1) C2

PART 3

A — Addendum for transport of animals by sea

(To be completed and attached to the veterinary certificate when transport to the European Community frontier includes, even for part of the journey, transportation by ship.)

B — Conditions for the authorisation of assembly centres

Approved assembly centres shall meet the following requirements:

I. They shall be supervised by an official veterinarian.

II. They shall each be situated at the centre of an area 20 km in diameter in which, according to official findings, for at least 30 days prior to their use as approved centres there has been no case of foot-and-mouth disease.

III. They shall, before each use as approved centres, be cleaned and disinfected with a disinfectant officially authorised in the exporting country as effective in the control of the disease mentioned in condition II above.

IV. They shall have, taking into account their animal capacity (a) a facility dedicated exclusively for this purpose; (b) appropriate facilities, easy to clean and disinfect, for loading, unloading and adequate housing of a suitable standard for the animals, for watering and feeding them, and for giving them any necessary treatment; (c) appropriate facilities for inspection and isolation; (d) appropriate equipment for cleaning and disinfecting rooms and trucks; (e) an appropriate storage area for fodder, litter and manure; (f) appropriate systems for collecting and disposal of waste water; (g) an office for the official veterinarian.

V. When operating, they shall have sufficient veterinarians to carry out all duties.

VI. They shall only admit animals that are individually identified so as to guarantee traceability. To this end, when animals are admitted the owner or person in charge of the centre shall ensure the animals are properly identified and accompanied by health documents or certificates for the species and categories involved. Moreover, this person shall record on a register or a database and retain for at least three years the name of the owner, the origin, date of entry and exit, number and identification of the animals or registration number of the herd of origin and their destination and the registration number of the carrier and the registration number of the lorry delivering or collecting animals from the premises.

VII. All animals passing through them shall fulfil the health conditions established for the importation of the relevant category of animal into the European Community.

VIII. Animals to be exported to the European Community which pass through an assembly centre must, within six days of arrival, be loaded and dispatched directly to the frontier of the exporting country: (a) without coming into contact with cloven-hoofed animals other than animals which fulfil the health conditions established for the importation of the relevant category of animal into the European Community; (b) segregated into consignments so that no consignment contains both animals for breeding or production and animals for immediate slaughter; (c) in transport vehicles or containers which have first been cleaned and disinfected with a disinfectant officially authorised in the exporting country as effective in the control of the disease mentioned in condition II above and which are so constructed that faeces, urine, litter or fodder cannot flow or fall out during transportation.

IX. Where the conditions for the export of animals to the Community require that a test be carried out within a specified period before loading, that period includes any period of assembly, up to six days, after the arrival of the animals at the approved centres.

X. The exporting country shall designate those approved centres which are approved for animals for breeding and production and those approved centres which are approved for animals for slaughter and shall notify the Commission and the competent central authorities of the Member States of the names and addresses of such premises and their regular updates.

XI. The exporting country shall determine the procedure for official supervision of approved centres and shall ensure that such supervision is carried out.

XII. They shall be regularly inspected in order to ascertain that the requirements for approval continue to be fulfilled. In case of failure and suspension, the approval may only be restored when the competent authority is satisfied the full compliance of the centre with all the provisions mentioned above.

C — Protocols for the standardisation of materials and testing procedures

Tuberculosis (TBL)

The single intradermal tuberculin test using bovine tuberculin shall be carried out according to Annex B to Directive 64/432/EEC. In the case of Suidae animals, the single intradermal tuberculin test using avian tuberculin shall be carried out according to Annex B to 64/432/EEC, except that the site of injection shall be the loose skin at the base of the ear.

Brucellosis (Brucella abortus) (BRL)

The serum agglutination test, complement fixation test, buffered brucella antigen test and enzyme linked immuno-absorbent assays tests (ELISA) shall be carried out according to Annex C to Directive 64/432/EEC.

Brucellosis (Brucella melitensis) (BRL)

Test shall be carried out according to Annex C to Directive 91/68/EEC.

Enzootic Bovine Leukosis (EBL)

The agar gel immuno-diffusion test and the enzyme linked immuno-absorbent assay test (ELISA) shall be carried out according to paragraphs A and C, chapter II of Annex D to Council Directive 64/432/EEC.

Bluetongue (BTG)

A. The blocking or competitive ELISA test shall be carried out according to the following protocol:

The competitive ELISA using monoclonal antibody 3-17-A3 is capable of identifying antibodies to all known serotypes of bluetongue virus (BTV).

The principle of the test is the interruption of the reaction between BTV antigen and a group-specific monoclonal antibody (3-17-A3) by the addition of test serum. Antibodies to BTV present in the test serum block the reactivity of the monoclonal antibody (Mab) and result in a reduction in the expected colour development after the addition of enzyme labelled anti-mouse antibody and chromogen/substrate. Sera can be tested at a single dilution of 1:5 (spot test — appendix 1) or may be titrated (serum titration — appendix 2) to give dilution end-point. Inhibition values higher than 50 % may be regarded as positive.

Material and reagents:

1. Appropriate ELISA microtitre plates.

2. Antigen: supplied as a cell extracted concentrate, prepared as described below, and stored at either -20 °C or -70 °C.

3. Blocking buffer: phosphate buffered saline (PBS) containing 0,3 % BTV negative adult bovine serum, 0,1 % (v/v) Tween-20 (supplied as polyoxyethylene sorbiton monolaurate syrup) in PBS.

4. Monoclonal antibody: 3-17-A3 (supplied as hybridoma tissue-culture supernatant) directed against the group-specific polypeptide VP7, stored at -20 °C or freeze-dried and diluted 1/100 with blocking buffer before use.

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

6. Chromogen and substrate: Orthophenylene diamine (OPD-chromogen) at a final concentration of 0,4 mg/ml in sterile distilled water. Hydrogen peroxide (30 % w/v-substrate) 0,05 % v/v added immediately before use (5μl H2O2 per 10 ml OPD). (Handle OPD with care — wear rubber gloves — suspected mutagen).

7. 1 Molar sulphuric acid: 26,6 ml of acid added to 473,4 ml of distilled water. (Remember — always add acid to water, never water to acid.)

8. Orbital shaker.

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

Test format

Cc: conjugate control (no serum/ no monoclonal antibody); C++: strong positive serum control; C+: weak positive serum control; C-: negative serum control; Cm: monoclonal antibody control (no serum).

Appendix 1: Spot dilution (1:5) format (40 sera/plate)

	Controls		Test sera
	1	2	3	4	5	6	7	8	9	10	11	12
A	Cc	C-	1	2	3	4	5	6	7	8	9	10
B	Cc	C-	1	2	3	4	5	6	7	8	9	10
C	C++	C++
D	C++	C++
E	C+	C+
F	C+	C+
G	Cm	Cm										40
H	Cm	Cm										40

Appendix 2: Serum titration format (10 sera/plate)

Controls

Test sera

C-

1:5

C-

1:10

C++

1:20

C++

1:40

C+

1:80

C+

1:160

1:320

1:640

Test protocol:

Conjugate control (Cc)

Wells 1A and 1B is a blank control consisting of BTV antigen and conjugate. This may be used to blank the ELISA reader.

Mab control (Cm)

Columns 1 and 2, rows G and H are the monoclonal antibody control and contain BTV antigen, monoclonal antibody and conjugate. These wells represent maximum colour. The mean of the optical density readings from this control represents the 0 % inhibition value.

Positive control (C++, C-)

Columns 1 and 2, rows C-D-E-F. These wells contain BTV antigen, BTV strong and weak positive antiserum respectively, Mab and conjugate.

Negative control (C-)

Wells 2A and 2B are the negative controls, which contain BTV antigen, BTV negative antiserum, Mab and conjugate.

Test sera

For large-scale serological surveys and rapid screening, sera could be tested at a single dilution of 1:5 (Appendix 1). Alternatively, 10 sera can be tested over a dilution range from 1:5 to 1:640 (Appendix 2). This will give some indication of the titre of antibody in the test sera.

Procedure:

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

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

3. Control wells: Add 100 μl of blocking buffer to Cc wells. Add 50 μl of positive and negative control sera, at a dilution of 1:5 (10 μl sera + 40 μl blocking buffer), to respective wells C-, C+ and C++. Add 50 μl blocking buffer to Mab control wells.

Spot titration method: Add a 1:5 dilution of each test serum in blocking buffer to duplicate wells of columns 3 to 12 (10 μl sera + 40 μl blocking buffer),

Serum titration method: Prepare a two-fold dilution series of each test sample (1:5 to 1:640) in blocking buffer across eight wells of single columns 3 to 12.

4. Immediately after the addition of the test sera, dilute Mab 1:100 in blocking buffer and add 50 μl to all wells of the plate except for the blank control.

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

6. Dilute rabbit anti-mouse concentrate to 1/5 000 in blocking buffer and add 50 μl to all wells of the plate.

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

8. Thaw the OPD and immediately before use add 5 μl of 30 % hydrogen peroxide to each 10 ml of OPD. Add 50 μl to all wells of the plate. Allow colour to develop for approximately 10 minutes and stop the reaction with 1 M sulphuric acid (50 μl per well). Colour should develop in the Mab control wells and in those wells containing sera with no antibody to BTV.

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

Analysis of results:

Using the software package print out the OD values, and the percentage inhibition (PI) for test and control sera based on the mean value recorded in the antigen control wells. The date 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 Mab control (antigen plus Mab in the absence of test sera) are between OD values 0,4 and 1,4. Any plate that fails to conform to the above criteria must be rejected.

If a computer software package is not available, print out the OD values using the ELISA printer. Calculate the mean OD value for the antigen control wells, which is equivalent to the 100 % value. Determine the 50 % OD value and manually calculate the positivity and negativity of each sample.

Percentage inhibition (PI) value = 100 — (OD of each test control/Mean OD of Cm) × 100.

The duplicate negative control serum wells and the duplicate blank wells should record PI values between +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 is developing. The strong and weak positive control sera should record PI values between +81 % and +100 %, and between +51 % and +80 %, respectively.

The diagnostic threshold for test sera is 50 % (PI 50 % or OD 50 %). Samples recording PI values > 50 % are recorded negative. Samples that record PI values above and below the threshold for the duplicate wells are considered doubtful; such samples may be retested in the spot test and/or titration. Positive samples may also be titrated to provide an indication of the degree of positivity.

Visual reading: Positive and negative samples are easily discernible by eye; weakly positive or strong negative samples may be more difficult to interpret by eye.

Preparation of BTV ELISA antigen:

1. Wash 40-60 roux of confluent BHK-21 cells three times with serum-free Eagle's medium and infect with bluetongue virus serotype 1 in serum-free Eagle's medium.

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

3. When CPE are complete in 90 to 100 % of the cell sheet of each roux, harvest the virus by shaking any still-attached cells from the glass.

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

5. Discard the supernatant and re-suspend the cells in approximately 30 ml of PBS containing 1 % ‘Sarkosyl’ and 2 ml phenylmethylsulphonyl fluoride (lysis buffer). This may cause the cells to form a gel and more lysis buffer may be added to reduce this effect. (NB: phenylmethylsulphonyl fluoride is harmful — handle with extreme caution.)

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

7. Centrifuge at 10 000 rpm for 10 minutes.

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

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

10. Pool the supernatants and centrifuge at 24 000 rpm (100 000 g) for 120 minutes at +4 °C over a 5 ml cushion of 40 % sucrose (w/v in PBS) using 30 ml Beckmann centrifuge tubes and an SW 28 rotor.

11. Discard the supernatant, drain the tubes thoroughly and re-suspend the pellet in PBS by sonication. Store the antigen in aliquots at -20 °C.

Titration of BTV ELISA antigen:

Bluetongue ELISA antigen is titrated by the indirect ELISA. Twofold dilutions of antigen are titrated against a constant dilution (1/100) monoclonal antibody 3-17-A3. The protocol is as follows:

1. Titrate a 1:20 dilution of BTV antigen in PBS across the microtitre plate in a twofold dilution series (50 μl/well) using a multichannel pipette.

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

3. Wash plates three times with PBS.

4. Add 50 μl of monoclonal antibody 3-17-A3 (diluted 1/100) to each well of the microtitre plate.

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

6. Wash plates three times with PBS.

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

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

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

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

B. The agar gel immuno-diffusion test shall be carried out according to the following protocol:

Antigen:

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

Known positive control serum:

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

Test serum

Procedure

1 % agarose prepared in borate or sodium barbitol buffer, pH 8,5 to 9,0, is poured into a petri dish to a minimum depth of 3,0 mm. A test pattern of seven moisture-free wells, each 5,0 mm in diameter, is cut in the agar. The pattern consists of one centre well and six wells arranged round it in a circle of radius 3 cm. The central well is filled with the standard antigen. Peripheral wells 2, 4 and 6 are filled with known positive serum, wells 1, 3 and 5 are filled with test sera. The system is incubated for up to 72 hours at room temperature in a closed humid chamber.

Interpretation

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

Epizootic haemorrhagic disease (EHD)

The agar gel immuno-diffusion test shall be carried out according to the following protocol:

Antigen:

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

Known positive control serum:

Test serum

Procedure

Interpretation

Infectious bovine rhinotracheitis (IBR)/infectious pustular vulvo-vaginitis (IPV)

A. The serum neutralisation test shall be carried out according to the following protocol:

Serum

All sera are heat-inactivated at 56 °C for 30 minutes before use.

Procedure

The constant virus-varying serum neutralisation test on microtitre plates employs MDBK or other susceptible cells. The Colorado, Oxford or any other reference strain of the virus is used at 100 TCID50 per 0,025 ml; inactivated undiluted serum samples are mixed with an equal volume (0,025 ml) of virus suspension. The virus/serum mixtures are incubated for 24 hours at 37 °C in the microtitre plates before the MDBK cells are added. Cells are used at a concentration which forms a complete monolayer after 24 hours.

Controls

(i) virus infectivity assay, (ii) serum toxicity controls, (iii) uninoculated cell culture controls, (iv) reference antisera.

Interpretation

The results of the neutralisation test and the titre of the virus used in the test are recorded after three to six days incubation at 37 °C. Serum titres are considered negative if there is no neutralisation at a dilution of 1/2 (undiluted serum).

B. Any other test recognised in the frame of Commission Decision 93/42/EC concerning additional guarantees to infectious rhinotracheitis for bovines destined for Member States or regions thereof free from the disease.

Foot-and-mouth disease (FMD)

A. Collecting oesophageal/pharyngeal samples and testing shall be carried out according to the following protocol:

Reagents

Prior to sampling, transport medium is prepared. Two ml volumes are dispensed in as many containers as there are animals to be sampled. The containers used should withstand freezing over solid CO2 or liquid nitrogen. Samples are obtained by the use of a specially-designed sputum collector or ‘probang’. To obtain a sample the probang cup is passed through the mouth, over the dorsum of the tongue and down into the upper part of the oesophagus. Attempts are made to scrape the surface epithelium of the upper oesophagus and pharynx by movements directed laterally and dorsally. The probang is then withdrawn, preferably after the animal has swallowed. The cup should be full and contain a mixture of mucus, saliva, oesophageal fluid and cellular debris. Care should be taken to ensure that each specimen contains some visible cellular material. Very rough handling which causes bleeding should be avoided. Samples from some animals may be heavily contaminated with ruminal contents. Such samples should be discarded and the mouth of the animal flushed with water, or preferably physiological saline, before repeat sampling.

Treatment of samples

Each sample collected in the probang cup is examined for quality and 2 ml added to an equal volume of transport medium in a container which can withstand freezing. The containers are tightly closed, sealed, disinfected and labelled. The samples are kept cool (+4 °C) and examined within three to four hours or placed over dry ice (-69 °C) or liquid nitrogen and kept frozen until examined. Between animals the probang is disinfected and washed in three changes of clean water.

Testing for FMD virus

Samples are inoculated into cultures of primary bovine thyroid cell cultures using at least three tubes per sample. Other susceptible cells e. g. primary bovine or porcine kidney cells can be used but it should be kept in mind that for some strains of FMD virus they are less sensitive. The tubes are incubated at 37 °C on a roller apparatus and examined daily for 48 hours for the presence of a cytopathic effect (CPE). If negative, cultures are blind passaged onto new cultures and re-examined for 48 hours. The specificity of any CPE must be confirmed.

Recommended transport media:

1. 0,08M phosphate buffer pH 7,2 containing 0,01 % bovine serum albumin, 0,002 % phenol red and antibiotics.

2. Tissue culture medium (e.g. Eagle's MEM) containing 0,04M Hepes buffer, 0,01 % bovine serum albumin and antibiotics, pH 7,2.

3. Antibiotics (per ml final) should be added to the transport medium, e.g. penicillin 1 000 IU, neomycin sulphate 100 IU, polymyxin B sulphate 50 IU, mycostatin 100 IU.

B. The virus neutralisation test shall be carried out according to the following protocol:

Reagents

Stock FMDV antigen is prepared in cell cultures or on cattle tongues and stored at -70 °C or less or at -20 °C after the addition of 50 % glycerol. This is the stock antigen. FMDV is stable under these conditions and titres vary little over a period of months.

Procedure

The test is carried out in flat-bottomed tissue culture grade microtitre plates using susceptible cells such as IB-RS-2, BHK-21 or calf kidney cells. Sera for the test are diluted 1/4 in serum-free cell culture medium with the addition of 100 IU/ml neomycin or other suitable antibiotics. Sera are inactivated at 56 °C for 30 minutes and 0,05 ml amounts are used to prepare a twofold series on microtitre plates using 0,05 ml diluting loops. Pretitrated virus also diluted in serum-free culture medium and containing 100 TCID50/0,05 ml is then added to each well. Following incubation at 37 °C for one hour to allow neutralisation to take place, 0,05 ml of suspension cells containing 0,5 to 1,0 × 106 cells per 1 ml in cell culture medium containing serum free of FMD antibody is added to each well and the plates are sealed. Plates are incubated at 37 °C. Monolayers are normally confluent within 24 hours. CPE is usually sufficiently advanced at 48 hours for a microscopic reading of the test. At this time a final microscopic reading may be made or the plates may be fixed and stained for macroscopic reading, for instance using 10 % formol-saline and 0,05 % methylene blue.

Controls

Controls in each test include homologous antiserum of known titre, a cell control, a serum toxicity control a medium control and a virus titration from which the actual amount of virus in the test is calculated.

Interpretation

Wells with evidence of CPE are considered to be infected and neutralisation titres are expressed as the reciprocal of the final dilution of serum present in the serum/virus mixtures at the 50 % end point estimated according to the Spearman-Karber method. (Karber, G., 1931, Archiv fuer Experimentelle Pathologie und Pharmokologie, 162, 480). Tests are considered to be valid when the actual amount of virus used per well in the test is between 101,5 and 102,5 TCID50 and when the titre of the reference serum is within twofold of its expected titre, estimated from the mode of previous titrations. When the controls are outside these limits the tests are repeated. An end point titre of 1/11 or less is taken as negative.

C. The detection and quantification of antibody by ELISA shall be carried out according to the following protocol:

Reagents

Rabbit antisera to 146S antigen of seven types of foot-and-mouth disease virus (FMDV) used at a predetermined optimum concentration in carbonate/bicarbonate buffer, pH 9,6. Antigens are prepared from selected strains of virus grown on monolayers of BHK-21 cells. The unpurified supernatants are used and pretitrated according to the protocol but without serum, to give a dilution which after the addition of an equal volume of PBST (phosphate buffered saline containing 0,05 % Tween-20 and phenol red indicator) would give an optical density reading of between 1,2 and 1,5. The viruses can be used inactivated. PBST is used as a diluent. Guinea-pig antisera are prepared by inoculating guinea pigs with 146S antigen of each serotype. A predetermined optimum concentration is prepared in PBST containing 10 % normal bovine serum and 5 % normal rabbit serum. Rabbit anti-guinea-pig immunoglobulin conjugated to horseradish peroxidase is used at a predetermined optimum concentration in PBST containing 10 % normal bovine serum and 5 % normal rabbit serum. Test sera are diluted in PBST.

Procedure:

1. ELISA plates are coated with 50 μl of rabbit antiviral sera overnight in a humidity chamber at room temperature.

2. Fifty microlitres of a duplicate, twofold series of each test serum starting at 1/4 are prepared in U-bottomed multiwell plates (carrier plates). Fifty microlitres of a constant dose of antigen are added to each well and the mixtures are left overnight at 4 °C. The addition of the antigen reduces the starting serum dilution to 1/8.

3. The ELISA plates are washed five times with PBST.

4. Fifty microlitres of serum/antigen mixtures are then transferred from the carrier plates to the rabbit-serum-coated ELISA plates and incubated at 37 °C for one hour on a rotary shaker.

5. After washing, 50 μl of guinea-pig antiserum to the antigen used in point 4 is added to each well. The plates are incubated at 37 °C for one hour a rotary shaker.

6. The plates are washed and 50 μl of rabbit anti-guinea-pig immunoglobulin conjugated to horseradish peroxidase is added to each well. The plates are incubated at 37 °C for one hour on a rotary shaker.

7. The plates are washed and 50 μl of orthophenylene diamine containing 0,05 % H2O2 (30 %) w/v is added to each well.

8. The reaction is stopped after 15 minutes with 1,25M H2SO4.

The plates are read spectrophotometrically at 492 nm on an ELISA reader linked to a microcomputer.

Controls

For each antigen used 40 wells contain no serum but contain antigen diluted in PBST. A duplicated twofold dilution series of homologous bovine reference antiserum. A duplicate twofold dilution series of negative bovine serum.

Interpretation

Antibody titres are expressed as the final dilution of tests serum giving 50 % of the mean OD value recorded in the virus control wells where test serum is absent. Titres in excess of 1/40 are considered positive.

References

Hamblin C, Barnett ITR and Hedger RS (1986) ‘A new enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus. I. Development and method of ELISA.’Journal of Immunological Methods, 93, 115 to 121.11.

Aujeszky's disease (AJD)

A. The serum neutralisation test shall be carried out according to the following protocol:

Serum

All sera are heat-inactivated at 56 °C for 30 minutes before use.

Procedure

The constant virus-varying serum neutralisation test on microtitre plates employs Vero or other sensitive cell systems. Aujeszky's disease virus is used at 100 TCID50 per 0,025 ml; inactivated undiluted serum samples are mixed with an equal volume (0,025 ml) of virus suspension. The virus/serum mixtures are incubated for two hours at 37 °C in the microtitre plates before the appropriate cells are added. Cells are used at a concentration which forms a complete monolayer after 24 hours.

Controls

(i) virus infectivity assay, (ii) serum toxicity controls, (iii) uninoculated cell culture controls, (iv) reference antisera.

Interpretation

The results of the neutralisation test and the titre of the virus used in the test are recorded after three to seven days incubation at 37 °C. Serum titres less than 1/2 (undiluted serum) are considered negative.

B. Any other test recognised in the frame of Commission Decision 2001/618/EC concerning additional guarantees to Aujeszky's disease for pigs destined for certain parts of the territory of the Community.

Transmissible gastroenteritis (TGE)

The serum neutralisation test shall be carried out according to the following protocol:

Serum

All sera are heat-inactivated at 56 °C for 30 minutes before use.

Procedure

The constant virus-varying serum neutralisation test on microtitre plates employs A72 (dog tumour) cells or other sensitive cell systems. TGE virus is used at 100 TCID50 per 0,025 ml; inactivated undiluted serum samples are mixed with an equal volume (0,025 ml) of virus suspension. The virus/serum mixtures are incubated for 30 to 60 minutes at 37 °C in the microtitre plates before the appropriate cells are added. Cells are used at a concentration which forms a complete monolayer after 24 hours. Each cell receives 0,1 ml of cell suspension.

Controls

(i) virus infectivity assay, (ii) serum toxicity controls, (iii) uninoculated cell culture controls, (iv) reference antisera.

Interpretation

The results of the neutralisation test and the titre of the virus used in the test are recorded after three to five days incubation at 37 °C. Serum titres less than 1/2 (final dilution) are considered negative. If undiluted serum samples are toxic to the tissue cultures, these sera may be diluted 1/2 before being used in the test. This will be equivalent to 1/4 final dilution of serum. Serum titres of less than 1/4 (final dilution) are considered negative in these cases.

Swine vesicular disease (SVD)

Tests for swine vesicular disease (SVD) shall be carried out according to Commission Decision 2000/428/EC.

Classical swine fever (CSF)

Tests for classical swine fever (CSF) shall be carried out according to Commission Decision 2002/106/EC.

The performance of tests for CSF should follow the guidelines set out in the OIE Manual of Standards for Diagnostic Tests and Vaccines — Chapter 2.1.13.

The sensitivity and specificity of the serological test for CSF should be carried out by a national laboratory with a quality assurance scheme in place. Tests employed must be shown to recognise a range of weak and strong positive reference sera and allow detection of antibody in early phase and convalescence.

▼C2

PART 4

Animal species

Taxon
ORDER	FAMILY	GENUS AND SPECIES
Artiodactila	Camelidae	Camelus ssp., Lama ssp., Vicugna ssp.

▼C2

Animal health conditions

Import and quarantine conditions for animals imported into St. Pierre and Miquelon within a period of less than six months prior to export to the European Community

Chapter 1

Residence and quarantine

1. Animals imported into St Pierre and Miquelon must reside in an authorised quarantine station for a minimum period of 60 days preceding export to the European Community. This period may be increased due to testing requirements for individual species. In addition the animals must comply with the following requirements:

(a) Separate consignments may enter the quarantine station. However, upon entry in the quarantine station all animals of the same species should be considered as a single group, and referred to as such. The quarantine period would commence for the whole group at the point that the last animal entered the facility.

(b) Within the quarantine station each specific group of animals must be maintained in isolation, with no direct or indirect contact with any other animals, including those from other consignments that may be present. Each consignment must be kept in the approved quarantine station and protected from vector insects.

(c) If, during the period of quarantine, the isolation of a group of animals is not maintained and contact is made with other animals, the quarantine is considered null and void, and the group must begin a new period of quarantine of the same time period as initially prescribed on entry into the quarantine station.

(d) animals to be exported to the European Community which pass through the quarantine station must be loaded and dispatched directly to the European Community:

(i) without coming into contact with animals other than animals which fulfil the health conditions established for the importation of the relevant category of animal into the European Community;

(ii) segregated into consignments so that no consignment can came in contact with animals not eligible for importation into the European Community;

(iii) in transport vehicles or containers which have first been cleansed and disinfected with a disinfectant officially authorized in St. Pierre and Miquelon as effective in the control of the diseases mentioned in Chapter II below and which are so constructed that faeces, urine, litter or fodder cannot flow or fall out of the vehicle during transportation.

2. The quarantine premises must at least meet the minimum standards laid down in Annex B to Directive 91/496/EEC, and the following conditions:

(a) they shall be supervised by an official veterinarian.

(b) they shall be situated at the centre of an area 20 km in diameter in which, according to official findings, for at least 30 days prior to their use as quarantine station there has been no case of foot-and-mouth disease.

(c) they shall, before being used as quarantine station, be cleansed and disinfected with a disinfectant officially authorized in St Pierre et Miquelon as effective in the control of the diseases mentioned in Chapter II.

(d) they shall operate, taking into account their animal capacity:

(i) a facility dedicated exclusively for this purpose, including adequate housing to a suitable standard for the animals;

(ii) appropriate facilities, that

— are easy to completely clean and disinfect,

— include facilities for safe loading and unloading,

— are able to fulfil all watering and feeding requirements for the animals,

— allow any necessary veterinary treatment to be easily administered;

(iii) appropriate facilities for inspection and isolation;

(iv) appropriate equipment for cleaning and disinfecting rooms and transport vehicles;

(v) an appropriate storage area for fodder, litter and manure;

(vi) an appropriate system for collecting waste water;

(vii) an office for the official veterinarian.

(e) when operating, they shall have sufficient veterinarians to carry out all duties,

(f) they shall only admit animals that are individually identified so as to guarantee traceability. To this end, when animals are admitted the owner or person in charge of the quarantine station shall ensure the animals are properly identified and accompanied by health documents or certificates for the species and categories involved. Moreover, this person shall record on a register or a data base, and retain for at least 3 years, the name of the owner, the origin, date of entry and exit, number and identification of the animals and their destination,

(g) the competent authority shall determine the procedure for official supervision of the quarantine station and shall ensure that such supervision is carried out; this supervision shall include regular inspections in order to ascertain that the requirements for approval continue to be fulfilled. In case of failure and suspension, the approval may only be restored when the competent authority is satisfied that the quarantine premises are in full compliance with all the provisions mentioned above.

Chapter 2

Animal health tests

1. General requirements

The animals must be subjected to the following tests carried out on samples of blood taken, if not specified otherwise, not earlier than 21 days after the commencing of the isolation period. The laboratory tests must be carried out in an approved laboratory in the European Community and all laboratory test and their results, vaccinations and treatments must be enclosed with the health certificate. In order to keep animal interventions to a minimum, sampling, tests and any vaccinations must be grouped as far as is possible whilst respecting the minimum time intervals required by the testing protocols.

2. Specific requirements

2.1. Camelids

2.1.1. Tuberculosis

(a) Test to be used: comparative intradermal reaction test using Bovine PPD and Avian PPD conforming to the standards for the manufacture of bovine and avian tuberculins as described in Annex B of Council Directive 64/432/EEC. The test has to be executed in the area behind the shoulder (axillary region) following the technique described in Annex B of Council Directive 64/432/EEC.

(b) Timing: the animals have to be tested within two days from their arrival in the quarantine station and after 42 days from the first test.

the reaction has to be considered:

— negative if the increased skin thickness is less than 2 mm,

— positive if the increased skin thickness is more than 4 mm,

— inconclusive if the increased skin thickness to the bovine PPD is between 2mm and 4 mm, or more than 4 mm but less then the reaction to the avian PPD.

(d) Options for action following testing:

If an animal presents a positive result to the intradermal-reaction to the bovine PPD, this animal shall be excluded from the group and the other animals have to be re-tested starting at least 42 days after the first positive test was administered: this has to be considered as the first test described in b).

If more than one animal of the group presents a positive result, the whole group shall to be rejected for exportation to the EC.

If one or more animals of the same group present an inconclusive reaction, the whole group will be re-tested after 42 days considering it as the first test described in b).

2.1.2. Brucellosis.

(a) Test to be used:

— B. Abortus: SAT and RBT as described respectively in point 2.6 and (2.5) in Annex C to Directive 64/432/EEC. In case of positive result, a Complement fixation test has to be performed for confirmation.

— B. Melitensis: SAT and RBT as described respectively in point 2.6 and 2.5 in Annex C to Directive 64/432/EEC. In case of positive result, a Complement fixation test following the method described in Annex C to Directive 91/68/EC has to be performed for confirmation.

— B. Ovis: Complement fixation test as described in Annex D to Directive 91/68/EC

(b) Timing: the animals have to be tested within two days from their arrival in the quarantine station and after 42 days from the first test.

A positive reaction to the tests will be as defined in Annex C to Directive 64/432/EEC.

(d) Options for action following testing:

Animals tested positive to one of the tests shall be excluded from the group and the other animals have to be re-tested starting at least 42 days after the first positive test was performed: this has to be considered as the first test described in (b).

Only the animals that tested negative to two consecutive tests performed as described in (b) shall be allowed for exportation to the EC.

2.1.3. Bluetongue and epizootic haemorrhagic disease (EHD).

(a) Test to be used: AGID test as described in Part 3 (C) of Annex I to Council Decision 79/542/EEC.

In case of positive reaction the animals have to be tested with Competitive ELISA test as described in Part 3(C) of Annex I to Council Decision 79/542/EEC to discriminate between the two diseases.

(b) Timing:

The animals have to be tested with negative result to two tests: the first within two days from their arrival in the quarantine station and the second after at least 21 days from the first test.

(i) Bluetongue

If one or more animals tested positive to the ELISA as described in Part 3 (C) of Annex I to Decision 79/542/EEC, the positive animal/animals shall be excluded from the group, and the whole remaining group will be quarantined for 100 days starting from the date in which the samples for the positive test were collected. The group can only be considered free of disease if regular checks by official veterinarians through the duration of the quarantine period fail to reveal clinical symptoms of disease, and the quarantine station remains free of Blue Tongue vectors (Culicoides).

If a further animal presents clinical symptoms of disease during the quarantine period as described above, the whole group shall be rejected for exportation to the EC.

(ii) Epizootic haemorrhagic disease (EHD).

If one or more animals tested positive reveals presence of antibodies to the EHD virus during confirmatory ELISA testing, the animal(s) shall be considered positive and shall be excluded from the group, and the whole group must be subject to repeat testing beginning at least 21 days after the initial positive diagnosis and again at least 21 days subsequently, both with negative results. If any additional animals are tested positive during repeat testing, the whole group shall be rejected for exportation to the EC.

2.1.4. Foot-and-mouth disease (FMD)

(a) Test to be used: Diagnostic tests (probang and serology) using ELISA and NV techniques under the protocols described in Part 3 (C) of Annex I to Decision 79/542/EEC.

(b) Timing: the animals have to be tested with negative results to two tests: the first within two days from their arrival in the quarantine station and the second after at least 42 days from the first test.

(c) Options for action following testing: If any animal tests positive for FMD virus, then all animals present in the quarantine station are not considered eligible for entry into the EC.

Note: Any detection of antibodies to structural or not structural proteins of FMD virus will be considered as a result of previous infection of FMD irrespective of the vaccination status.

2.1.5. Rinderpest

(a) Test to be used: The competitive ELISA test as described in the OIE manual is the prescribed test for international trade and is test of choice. Serum neutralisation test, or other recognised tests in accordance with the protocols described in relevant sections of the OIE manual can also be used.

(b) Timing: the animals have to be tested twice: the first within two days from their arrival in the quarantine station and the second after at least 42 days from the first test.

(c) Options for action following testing: If any animal tests positive for Rinderpest virus, then all animals present in the quarantine station are not considered eligible for entry into the EC.

2.1.6. Vesicular stomatitis

(a) Test to be used: ELISA, virus neutralisation test, or other recognised test in accordance with the protocols described in relevant sections of the OIE manual.

(b) Timing: the animals have to be tested twice: the first within two days from their arrival in the quarantine station and the second after at least 42 days from the first test.

(c) Options for action following testing: If any animal tests positive for Vesicular Stomatitis virus, then all animals present in the quarantine station are not considered eligible for entry into the EC.

2.1.7. Rift Valley Fever

(a) Test to be used: ELISA, virus neutralisation test, or other recognised test in accordance with the protocols described in relevant sections of the OIE manual.

(b) Timing: the animals have to be tested twice: the first within two days from their arrival in the quarantine station and the second after at least 42 days from the first test.

(c) Options for action following testing: If any animal displays evidence of exposure to Rift Valley Fever agent, then all animals present in the quarantine station are not considered eligible for entry into the EC.

2.1.8. Lumpy skin disease

(a) Test to be used: Serology using ELISA, virus neutralisation test, or other recognised test in accordance with the protocols described in relevant sections of the OIE manual.

(b) Timing: the animals have to be tested twice: the first within two days from their arrival in the quarantine station and the second after at least 42 days from the first test.

(c) Options for action following testing: If any animal displays evidence of exposure to Lumpy Skin Disease, the whole group shall be rejected for exportation to the EC.

2.1.9 Crimean Congo haemorrhagic fever

(a) Test to be used: ELISA, virus neutralisation test, Immunofluorescence test or other recognised test.

(b) Timing: the animals have to be tested twice: the first within two days from their arrival in the quarantine station and the second after at least 42 days from the first test.

(c) Options for action following testing: If any animal displays evidence of exposure to Crimean Congo Haemorrhagic fever agent, then that animal shall be excluded from the group.

2.1.10. Surra (Trypanosoma evansi)

(a) Test to be used: The parasitic agent can be identified in concentrated blood samples in accordance with the protocols described in relevant sections of the OIE manual.

(b) Timing: the animals have to be tested twice: the first within two days from their arrival in the quarantine station and the second after at least 42 days from the first test.

(c) Options for action following testing: If T. evansi is detected in any animal, then that animal shall be excluded from the group. The remaining group should then undergo internal and external antiparasitic treatment using suitable agents that are effective against T. evansi.

2.1.11. Malignant catarrhal fever

(a) Test to be used: Detection of viral DNA is the preferred method, based on identification by immunofluorescence or immunocytochemistry using the protocols described in relevant sections of the OIE manual.

(b) Timing: the animals have to be tested twice: the first within two days from their arrival in the quarantine station and the second after at least 42 days from the first test.

(c) Options for action following testing: If any animal displays evidence of exposure to MCF, then the whole group shall be rejected for exportation to the EC.

2.1.12. Rabies

Vaccination: Rabies vaccination may be carried out in certain cases and the animal should be blood sampled and a serum neutralisation test for antibodies carried out.

2.1.13. Bovine leucosis. (only in the case the animals are destined to a free region)

(a) Test to be used: AGID or blocking ELISA, in accordance with the protocols described in the OIE manual.

(b) Timing: the animals have to be tested twice: the first within two days from their arrival in the quarantine station and the second after at least 42 days from the first test.

(c) Options for action following testing: animals tested positive to the test shall be excluded from the group and the other animals have to be re-tested starting at least 21 days after the first positive test was performed: this has to be considered as the first test described in (b).

Only the animals that tested negative to two consecutive tests performed as described in (b) shall be allowed for exportation to the EC.

ANNEX II

FRESH MEAT

PART 1

List of third countries or parts thereof (1)

Country	Code of Territory	Description of territory	Veterinary certificate		Specific conditions
Country	Code of Territory	Description of territory	Model(s)	SG	Specific conditions
1	2	3	4	5	6
AL — Albania	AL-0	Whole country	—
AR — Argentina	AR-0	Whole country	EQU
	AR-1	The Provinces of Buenos Aires, Catamarca, Corrientes, Entre Ríos, La Rioja, Mendoza, Misiones, Neuquen, Rio Negro, San Juan, San Luis, Santa Fe, and Tucuman.	BOV	A	1 and 2
	AR-2	La Pampa and Santiago del Estero	BOV	A	1 and 2
	AR-3	Cordoba	BOV	A	1 and 2
	AR-4	Chubut, Santa Cruz and Tierra del Fuego	BOV, OVI
	AR-5	Formosa (only the territory of Ramon Lista) and Salta (only the department of Rivadavia)	BOV	A	1 and 2
	AR-6	Salta (only the departments of General Jose de San Martin, Oran, Iruya, and Santa Victoria)	BOV	A	1 and 2
	AR-7	Chaco, Formosa (except the territory of Ramon Lista), Salta (except the departments of General Jose de San Martin, Rivadavia, Oran, Iruya, and Santa Victoria), Jujuy	BOV	A	1 and 2
AU — Australia	AU-0	Whole country	BOV, OVI, POR, EQU, RUF, RUW, SUF, SUW
BA — Bosnia Hercegovina	BA-0	Whole country	—
BG — Bulgaria	BG-0	Whole country	EQU
	BG-1	The provinces of Varna, Dobrich, Silistra, Choumen, Targovitchte, Razgrad, Rousse, V.Tarnovo, Gabrovo, Pleven, Lovetch, Plovdic, Smolian, Pasardjik, Sofia district, Sofia city, Pernik, Kustendil, Blagoevgrad, Vratza, Montana and Vidin	BOV, OVI RUW, RUF
	BG-2	The provinces of Bourgas, Jambol, Sliven, Starazagora, Hasskovo, Kardjaliand and the 20 km-wide corridor on the border with Turkey	—
BH — Bahrain	BH-0	Whole country	—
BR — Brazil	BR-0	Whole country	EQU
	BR-1	States of Paraná, Minas Gerais (except regional delegations of Oliveira, Passos, São Gonçalo de Sapucai, Setelagoas and Bambuí), São Paulo, Espíritu Santo, Mato Grosso do Sul (except for the municipalities of Sete Quedas, Sonora, Aquidauana, Bodoqueno, Bonito, Caracol, Coxim, Jardim, Ladario, Miranda, Pedro Gomes, Porto Murtinho, Rio Negro, Rio Verde of Mato Grosso and Corumbá), Santa Catarina, Goias and the regional units of Cuiaba (except for the municipalities of San Antonio de Leverger, Nossa Senhora do Livramento, Pocone and Barão de Melgaço), Caceres (except for the municipality of Caceres), Lucas do Rio Verde, Rondonopolis (except for the municipality of Itiquiora), Barra do Garça and Barra do Burges in Mato Grosso.	BOV	A	1 and 2
	BR-2	State of Rio Grande do Sul	BOV	A	1 and 2
	BR-3	State of Mato Grosso do Sul, municipality of Sete Quedas	BOV	A	1 and 2
BW — Botswana	BW-0	Whole country	EQU, EQW
	BW-1	The veterinary disease control zones 5, 6, 7, 8, 9 and 18	BOV, OVI, RUF, RUW	F	1 and 2
	BW-2	The veterinary disease control zones 10, 11, 12, 13 and 14	BOV, OVI, RUF, RUW	F	1 and 2
BY — Belarus	BY-0	Whole country	—
BZ — Belize	BZ-0	Whole country	BOV, EQU
CA — Canada	CA-0	Whole country	BOV, OVI, POR, EQU, SUF, SUW, RUF, RUW	G
CH — Switzerland	CH-0	Whole country	BOV, OVI, POR, EQU, RUF, RUW, SUF, SUW
CL — Chile	CL-0	Whole country	BOV, OVI, POR, EQU, RUF, RUW, SUF
CN — China (People’s Republic of)	CN-0	Whole country	—
CO — Colombia	CO-0	Whole country	EQU
	CO-1	The zone included within the borderlines from the point where the Murri River flows into the Atrato River, downstream along the Atrato River to where it flows into the Atlantic Ocean from this point to the Panamanian border following the Atlantic coast-line to Cabo Tiburón; from this point to the Pacific Ocean following the Columbian-Panamanian border; from this point to the mouth of the Valle River along the Pacific coast and from this point along a straight line to the point where the Murri River flows into the Atrato River.	BOV	A	2
	CO-3	The zone included within the borderlines from the mouth of the Sinu River on the Atlantic Ocean, upstream along the Sinu River to its head-Waters of Alto Paramillo, from this point to Puerto Rey on the Atlantic Ocean, following the borderline between the Department of Antiquia and Córdoba, and from this point to the mouth of the Sinu River along the Atlantic coast.	BOV	A	2
CR — Costa Rica	CR-0	Whole country	BOV, EQU
CS — Serbia and Montenegro (2)	CS-0	Whole country	BOV, OVI, EQU
CU — Cuba	CU-0	Whole country	BOV, EQU
DZ — Algeria	DZ-0	Whole country	—
ET — Ethiopia	ET-0	Whole country	—
FK — Falkland Islands	FK-0	Whole country	BOV, OVI, EQU
GL — Greenland	GL-0	Whole country	BOV, OVI, EQU, RUF, RUW
GT — Guatemala	GT-0	Whole country	BOV, EQU
HK — Hong Kong	HK-0	Whole country	—
HN — Honduras	HN-0	Whole country	BOV, EQU
HR — Croatia	HR-0	Whole country	BOV, OVI, EQU, RUF, RUW
IL — Israel	IL-0	Whole country	—
IN — India	IN-0	Whole country	—
IS — Iceland	IS-0	Whole country	BOV, OVI, EQU, RUF, RUW
KE — Kenya	KE-0	Whole country	—
MA — Morocco	MA-0	Whole country	EQU
MG — Madagascar	MG-0	Whole country	—
(MK) — Former Yugoslav Republic of Macedonia (3)	MK-0	Whole country	OVI, EQU
MU — Mauritius	MU-0	Whole country	—
MX — Mexico	MX-0	Whole country	BOV, EQU
NA — Namibia	NA-0	Whole country	EQU, EQW
NA — Namibia	NA-1	South of the cordon fences which extend from Palgrave Point in the west to Gam in the east	BOV, OVI, RUF, RUW	F	2
NC — New Caledonia	NC-0	Whole country	BOV, RUF, RUW
NI — Nicaragua	NI-0	Whole country	—
NZ — New Zealand	NZ-0	Whole country	BOV, OVI, POR, EQU, RUF, RUW, SUF, SUW
PA — Panama	PA-0	Whole country	BOV, EQU
PY — Paraguay	PY-0	Whole country	EQU
PY — Paraguay	PY-1	Chaco central and San Pedro areas	BOV	A	1 and 2
RO — Romania	RO-0	Whole country	BOV, OVI, EQU, RUW, RUF
RU — Russian Federation	RU-0	Whole country	—
RU — Russian Federation	RU-1	Region of Murmansk (Murmanskaya oblast)	RUF
SV — El Salvador	SV-0	Whole country	—
SZ — Swaziland	SZ-0	Whole country	EQU, EQW
	SZ-1	Area west of the red line fences which extends northwards from the river Usutu to the frontier with South Africa west of Nkalashane,	BOV, RUF, RUW	F	2
	SZ-2	The veterinary foot and mouth surveillance and vaccination control areas as gazetted as a Statutory Instrument under legal notice number 51 of 2001	BOV, RUF, RUW	F	1 and 2
TH — Thailand	TH-0	Whole country	—
TN — Tunisia	TN-0	Whole country	—
TR — Turkey	TR-0	Whole country	—
TR — Turkey	TR-1	The provinces of Amasya, Ankara, Aydin, Balikesir, Bursa, Cankiri, Corum, Denizli, Izmir, Kastamonu, Kutahya, Manisa, Usak, Yozgat and Kirikkale	EQU
UA — Ukraine	UA-0	Whole country	—
US — United States of America	US-0	Whole country	BOV, OVI, POR, EQU, SUF, SUW, RUF, RUW	G
UY — Uruguay	UY-0	Whole country	EQU
			BOV	A	1
			OVI	A	1 and 2
ZA — South Africa	ZA-0	Whole country	EQU, EQW
ZA — South Africa	ZA-1	The whole country except: — the part of the foot-and-mouth disease control area situated in the veterinary regions of Mpumalanga and northern provinces, in the district of Ingwavuma of the veterinary region of Natal and in the border area with Botswana east of longitude 28°, and — the district of Camperdown, in the province of KwaZuluNatal	BOV, OVI, RUF, RUW	F	2
ZW — Zimbabwe	ZW-0	Whole country	—
(1) Without prejudice to specific certification requirements provided for by Community agreements with third countries. (2) Not including Kosovo as defined by United Nations Security Council Resolution 1244 of 10 June 1999. (3) Former Yugoslav Republic of Macedonia; provisional code that does not affect the definitive denomination of the country to be attributed after the conclusion of the negotiations currently taking place in the United Nations. — = No certificate laid down and fresh meat imports are prohibited.

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Specific conditions referred to in column 6

‘1’: Geographic and timing restrictions

Code of territory	Veterinary certificate		Time period/dates for which importation into the Community is authorised or not authorised in relation to dates of slaughter/killing of animals from which the meat was obtained
	Model	SG
AR-1	BOV	A	Before and including 31 January 2002	Not authorised
			After and including 1 February 2002	Authorised
AR-2	BOV	A	Before and including 8 March 2002	Not authorised
			After and including 9 March 2002	Authorised
AR-3	BOV	A	Before and including 26 March 2002	None
			After and including 27 March 2002	Authorised
AR-4	BOV, OVI, RUM, RUF	—	Before and including 28 February 2002	Not authorised
			After and including 1 March 2002	Authorised
AR-5	BOV	A	Before and including 10 July 2003	Authorised
			After and including 11 July 2003	Not Authorised
AR-6	BOV	A	Before and including 4 September 2003	Authorised
			After and including 5 September 2003	Not Authorised
AR-7	BOV	A	After 1 February 2002 and before and including 7 October 2003	Authorised
			After and including 8 October 2003	Not Authorised
BR-2	BOV	A	Before and including 30 November 2001	Not authorised
			After and including 1 December 2001	Authorised
BR-3	BOV	A	Before and including 31 October 2002	Authorised
			After and including 1 November 2002	Not authorised
BW-1	BOV, OVI, RUM, RUF	A	Before and including 7 July 2002	Not authorised
			After and including 8 July to 22 December 2002	Authorised
			After and including 23 December 2002 to 6 June 2003	Not authorised
			After and including 7 June 2003	Authorised
BW-2	BOV, OVI, RUM, RUF	A	Before and including 6 March 2002	Not authorised
			After and including 7 March 2002	Authorised
PY-1	BOV	A	Before and including 31 August 2002	Not authorised
			After and including 1 September 2002 to 19 February 2003	Authorised
			After and including 20 February 2003	Not authorised
SZ-2	BOV,RUF, RUW	A	Before and including 3 August 2003	Not authorised
			After and including 4 August 2003	Authorised
UY-0	BOV, OVI	A	Before and including 31 October 2001	Not authorised
			After and including 1 November 2001	Authorised

‘2’: Category restrictions:

No offal authorised (except bovine diaphragm and masseter muscles).

PART 2

Models of veterinary certificates

Model(s):

‘BOV’

Model of veterinary certificate for fresh meat of domestic bovine animals (Bos taurus, Bison bison, Bubalus bubalis and their cross-breeds)

‘POR’

Model of veterinary certificate for fresh meat of domestic porcine animals (Sus scrofa)

‘OVI’

Model of veterinary certificate for fresh meat of domestic sheep (Ovis aries) and goats (Capra hircus)

‘EQU’

Model of veterinary certificate for fresh meat of domestic equine animals (Equus caballus, Equus asinus and their cross-breeds)

‘RUF’

Model of veterinary certificate for fresh meat of farmed non-domestic animals other than suidae and solipeds

‘RUW’

Model of veterinary certificate for fresh meat of wild non-domestic animals other than suidae and solipeds

‘SUF’

Model of veterinary certificate for fresh meat of farmed non-domestic suidae

‘SUW’

Model of veterinary certificate for fresh meat of wild non-domestic suidae

‘EQW’

Model of veterinary certificate for fresh meat of wild non-domestic solipeds

SG (Supplementary guarantees):

guarantees regarding the maturation, pH measurement and boning of fresh meat, excluding offal, certified according to the models of certificates BOV (point 10.6), OVI (point 10.6), RUF (point 10.7) and RUW (point 10.4).

‘B’

guarantees regarding matured trimmed offal as described in the model of certificate BOV (point 10.6).

‘C’

guarantees regarding laboratory test for classical-swine-fever in the carcases from which fresh meat certified according to the model of certificate SUW (point 10.3 bis) was obtained.

‘D’

guarantees regarding swill feed on holding(s) of animals from which fresh meat certified according to models of certificate POR (point 10.3 d)) was obtained.

‘E’

guarantees regarding tuberculosis test in the animals from where fresh meat certified according to the model of certificate BOV (point 10.4 d)) was obtained.

‘F’

guarantees regarding the maturation and de-boning of fresh meat, excluding offal, certified according to the models of certificates BOV (point 10.6), OVI (point 10.6), RUF (point 10.7) and RUW (point 10.4).

‘G’

guarantees regarding 1, exclusion of offals and spinal cord; and 2, testing and origin of cervid animals in relation to chronic wasting disease as referred to in the models of certificates RUF (point 9.2.1) and RUW (point 9.3.1).

Notes

(a) Veterinary certificates shall be produced by the exporting country, based on the models appearing in Part 2 of Annex II, according to the layout of the model that corresponds to the meats concerned. They shall contain, in the numbered order that appears in the model, the attestations that are required for any third country and, as the case may be, those supplementary guarantees that are required for the exporting third country or part thereof.

(b) A separate and unique certificate must be provided for meat that is exported from a single territory appearing in columns 2 and 3 of Part 1 of Annex II which is consigned to the same destination and transported in the same railway wagon, lorry, aircraft or ship.

(d) It shall be drawn up in at least one of the official languages of the EU Member State in which the inspection at the border post shall be carried out and of the EU Member State of destination. However, these Member States may allow other languages, if necessary, accompanied by an official translation.

(e) If for reasons of identification of the items of the consignment (schedule in point 8.3 of the model of certificate), additional pages are attached to the certificate, these pages shall also be considered as forming part of the original of the certificate by the application of the signature and stamp of the certifying official veterinarian, in each of the pages.

(g) The original of the certificate must be completed and signed by an official veterinarian. In doing so, the competent authorities of the exporting country shall ensure that the principles of certification equivalent to those laid down in Council Directive 96/93/EC are followed.

The colour of the signature shall be different to that of the printing. The same rule applies to stamps other than those embossed or watermarked.

(h) The original of the certificate must accompany the consignment at the EU border inspection post.

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MODEL RUF

MODEL RUW