‘I’

:

for transit through the territory of a third country of live animals for immediate slaughter or live bovine animals for fattening which are consigned from a Member State and destined to another Member State in lorries which have been sealed with a serially numbered seal.

The seal number must be entered on the health certificate issued in accordance with the model laid down in Annex F to Directive 64/432/EEC (6) for live bovine animals for slaughter and fattening and in accordance with Model I of Annex E to Directive 91/68/EEC (7) for ovine and caprine animals for slaughter.

In addition, the seal must be intact on arrival at the designated border inspection post of entry into the Union and the seal number recorded in the integrated computerised veterinary system of the Union (TRACES).

The certificate must be stamped at the exit point of the Union by the competent veterinary authority prior to transiting one or more third countries, with the following wording ‘ONLY FOR TRANSIT BETWEEN DIFFERENT PARTS OF THE EUROPEAN UNION VIA THE FORMER YUGOSLAV REPUBLIC OF MACEDONIA/MONTENEGRO/SERBIA (8)  (9)’.

Bovine animals for fattening must be transported directly to the holding of destination designated by the competent veterinary authority of destination. Those animals must not be moved from that holding unless for immediate slaughter.

‘II’

:

territory recognised as having an official tuberculosis-free status for the purposes of exports to the Union of live 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 Union of live 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 Union of live 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 Union of live 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 Union of live 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 Union of live 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 Union of live 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 Union of live animals certified according to the model of certificate POR-X.

‘BOV-X’

:

Model of veterinary certificate for domestic bovine animals (including Bubalus and Bison species and their cross-breeds) intended for breeding and/or production after importation.

‘BOV-Y’

:

Model of veterinary certificate for domestic bovine animals (including Bubalus and Bison species and their cross-breeds) intended for immediate slaughter after importation.

‘OVI-X’

:

Model of veterinary certificate for domestic ovine animals (Ovis aries) and domestic caprine animals (Capra hircus) intended for breeding and/or production after importation.

‘OVI-Y’

:

Model of veterinary certificate for domestic ovine animals (Ovis aries) and domestic caprine animals (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 animals of the order Artiodactyla (excluding bovine animals (including Bubalus and Bison species and their cross-breeds), Ovis aries, Capra hircus, Suidae and Tayassuidae), and of the families Rhinocerotidae and Elephantidae.

‘SUI’

:

Model of veterinary certificate for non-domestic Suidae, Tayassuidae and Tapiridae.

‘CAM’

:

Model of specific attestation for animals imported from St Pierre and Miquelon under the conditions provided for in Part 7 of Annex I.

‘A’

:

guarantees regarding Bluetongue and Epizootic-haemorrhagic-disease tests on animals certified according to the model of certificate BOV-X (point II.2.8 B), OVI-X (point II.2.6 D) and RUM (point II.2.6).

‘B’

:

guarantees regarding Swine-vesicular-disease and Classical-swine-fever tests on animals certified according to the model of certificate POR-X (point II.2.4 B) and SUI (point II.2.4 B).

‘C’

:

guarantees regarding Brucellosis test on animals certified according to the model of certificate POR-X (point II.2.4 C) and SUI (point II.2.4 C).

I.

They must be supervised by an official veterinarian.

II.

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

III.

They must, before each use as approved assembly centres, be cleansed and disinfected with a disinfectant officially authorised in the exporting country as effective for the control of foot-and-mouth disease.

IV.

They must have, taking into account their animal capacity:

V.

When operating, they must have sufficient veterinarians to carry out all duties set out in Part 5;

VI.

They must only admit animals that are individually identified so as to guarantee traceability. To this end, when animals are admitted the owner or the person in charge of the centre must ensure that the animals are properly identified and accompanied by health documents or certificates for the species and categories involved.

In addition, the owner or the person in charge of the assembly centre must record on a register or in a data base, and retain for at least three years the name of the owner, the origin of the animals, the dates of entry and exit, the identification number of the animals or registration number of the herd of origin and the holding of destination, and, the registration number of the carrier and the registration number of the lorry delivering or collecting animals from that assembly centre.

VII.

All animals passing through the assembly centre must fulfil the health conditions established for the introduction of the relevant category of animal into the Union.

VIII.

Animals to be introduced into the Union which pass through an assembly centre must, within six days of arrival at the assembly centre, be loaded and dispatched directly to the border of the exporting country:

IX.

Where the conditions for the export of animals to the Union require that a test is carried out within a specified period before loading, that period must include any period of assembly, up to six days, from the date of arrival of the animals at the approved assembly centre.

X.

The exporting third country must designate the centres which are approved for animals for breeding and production and those centres which are approved for animals for slaughter and must notify the Commission and the competent central authorities of the Member States of the names and addresses of such premises. That information must be updated regularly.

XI.

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

XII.

The approved assembly centres must be regularly inspected by the competent authority of the third country in order to check that the requirements for approval set out in points I to XI continue to be fulfilled.

If those inspections show that those conditions are no longer complied with, the approval of the centre must be suspended. The approval may be restored only when the competent authority of the third country is satisfied that the centre fully complies with the conditions set out in points I to XI.

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 H2 O2 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 Acid must be added to water, never water to acid.)

8.

Orbital shaker.

9.

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

Conjugate control (Cc)

:

Wells 1A and 1B are 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 may be tested at a single dilution of 1:5 (Appendix 1). Alternatively, 10 sera may 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.

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 ul of positive and negative control sera, at a dilution of 1:5 (10 μ l sera + 40 μl blocking buffer), to respective wells C-, C+ and C++. Add 50μl 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),

or

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 O-Phenylenediamine dihydrochloride (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 Molar 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.

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 re-suspend 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.

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 pre-titrated 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).

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 must be examined against a dark background and using indirect illumination.

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 must be examined against a dark background and using indirect illumination.

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).

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 must 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, if possible after the animal has swallowed. The cup must be full and contain a mixture of mucus, saliva, oesophageal fluid and cellular debris. Care must be taken to ensure that each specimen contains some visible cellular material. Very rough handling which causes bleeding must be avoided. Samples from some animals may be heavily contaminated with ruminal contents. Such samples must be discarded and the mouth of the animal flushed with water, or preferably physiological saline, before repeat sampling.

Treatmentof 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 such as primary bovine or porcine kidney cells can be used but it must 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.

1.

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

2.

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

3.

Antibiotics (per ml final) must be added to the transport medium such as penicillin1 000 IU, neomycin sulphate100 IU, polymyxin B sulphate50 IU, mycostatin100 IU.

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. Pre-titrated 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.

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.

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.

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.

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.

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 is equivalent to 1/4 final dilution of serum. Serum titres of less than 1/4 (final dilution) are considered negative in these cases.

(a)

Separate consignments may enter the quarantine station. However, upon entry in the quarantine station all animals of the same species in the quarantine facility must be considered as a single group, and referred to as such. The quarantine period must commence for the whole group at the time when the last animal entered the quarantine 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 period must begin again for the same duration as initially prescribed on entry into the quarantine station.

(d)

Animals to be introduced into the Union which pass through the quarantine station must be loaded and dispatched directly to the Union:

(a)

they must be supervised by an official veterinarian;

(b)

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

(c)

they must, before being used as a quarantine station, be cleansed and disinfected with a disinfectant officially authorised in St Pierre et Miquelon as effective in the control of the diseases referred to in Chapter 2;

(d)

they must operate, taking into account their animal capacity:

(e)

when operating, they must have sufficient veterinarians to carry out all duties;

(f)

they must only admit animals that are individually identified so as to guarantee traceability. To this end, when animals are admitted the owner or the person in charge of the quarantine station must ensure that the animals are properly identified and accompanied by health certificates for the species and categories involved. In addition, the owner or the person in charge of the quarantine station must record on a register or in a data base, and retain for at least three years, the name of the owner, the origin of the animals in the consignment, the dates of entry and exit of the animals in the consignment, the identification number of the animals in the consignment and their place of destination;

(g)

the competent authority must determine the procedure for official supervision of the quarantine station and must ensure that such supervision is carried out; this supervision must 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 conditions set out in points (a) to (g).

(a)

Test to be used: comparative intradermal reaction test using Bovine purified protein derivative (PPD) and Avian PPD conforming to the standards for the manufacture of bovine and avian tuberculins as described in point 2.1.2 of Annex B of Directive 64/432/EEC.

The test must be executed in the area behind the shoulder (axillary region) following the technique described in point 2.2.4 of Annex B of Directive 64/432/EEC.

(b)

Timing: the animals must be tested within two days from the date of arrival in the quarantine station and 42 days from the date of the first test.

(c)

Interpretation of tests:

the reaction shall be considered:

(d)

Options for action following testing:

If an animal presents a positive result to the intradermal-reaction to the bovine PPD, that animal shall be excluded from the group and the other animals shall be re-tested starting at least 42 days from the date of the first positive test was administered and this shall 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 be rejected for exportation to the Union.

If one or more animals of the same group present an inconclusive reaction, the whole group shall be re-tested starting at least 42 days from the date of the first test was administered and it shall be considered as the first test described in (b).

(a)

Test to be used:

(b)

Timing: the animals have to be tested within two days from the date of their arrival in the quarantine station and 42 days from the date of the first test.

(c)

Interpretation of tests:

A positive reaction to the tests shall 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 shall be re-tested starting at least 42 days from the date the first positive test was performed: this shall 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 the introduction to the Union.

(a)

Test to be used: agar gel immunodiffusion (AGID) test as described in Part 6 of Annex I to Regulation (EU) No 206/2010 (SANCO/4787/2009).

In case of a positive reaction the animals shall be tested with competitive ELISA test as described in Part 6 of Annex I to Regulation (EU) No 206/2010 (SANCO/4787/2009) to discriminate between the two diseases.

(b)

Timing:

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

(c)

Options for action following testing:

(a)

Test to be used: Diagnostic tests (probang and serology) using ELISA and (Virus Neutralisation) (VN) techniques in accordance with the Protocols described in Part 6 of Annex I to Regulation (EU) No 206/2010 (SANCO/4787/2009).

(b)

Timing: the animals shall be tested with negative results to two tests: the first within two days from the date of their arrival in the quarantine station and the second at least 42 days from the date of the first test.

(c)

Options for action following testing: If any animal tests positive for the FMD virus, then none of the animals present in the quarantine station shall be considered as eligible for introduction into the Union.

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

(a)

Test to be used: The competitive ELISA test as described in the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, latest version, 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 may also be used.

(b)

Timing: the animals shall be tested twice: the first within two days from the date of their arrival in the quarantine station and the second at least 42 days from the date of the first test.

(c)

Options for action following testing: If any animal tests positive for the Rinderpest virus, then none of the animals present in the quarantine station shall be considered as eligible for introduction into the Union.

(a)

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

(b)

Timing: the animals shall be tested twice: the first within two days from the date of their arrival in the quarantine station and the second at least 42 days from the date of the first test.

(c)

Options for action following testing: If any animal tests positive for vesicular stomatitis virus, then none of the animals present in the quarantine station shall be considered as eligible for introduction into the Union.

(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 shall be tested twice: the first within two days from the date of their arrival in the quarantine station and the second at least 42 days from the date of the first test.

(c)

Options for action following testing: If any animal displays evidence of exposure to rift valley fever agent, then none of the animals present in the quarantine station shall be considered as eligible for introduction into the Union.

(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 shall be tested twice: the first within two days from the date of their arrival in the quarantine station and the second at least 42 days from the date of the first test.

(c)

Options for action following testing: If any animal displays evidence of exposure to lumpy skin disease, then none of the animals present in the quarantine station shall be considered as eligible for introduction into the Union.

(a)

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

(b)

Timing: the animals shall be tested twice: the first within two days from the date of their arrival in the quarantine station and the second at least 42 days from the date of the first test.

(c)

Options for action following testing: If any animal displays evidence of exposure to crimean congo haemorrhagic fever agent, then none of the animals present in the quarantine station shall be considered as eligible for introduction into the Union.

(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 shall be tested twice: the first within two days from the date of their arrival in the quarantine station and the second at least 42 days from the date of the first test.

(c)

Options for action following testing: If T. evansi is detected in any animal in the consignment, then that animal shall be considered not eligible for introduction into the Union. The remaining animals of the group shall then undergo internal and external antiparasitic treatment using suitable agents that are effective against T. evansi.

(a)

Test to be used: Detection of viral DNA based on identification by immunofluorescence or immunocytochemistry using the protocols described in relevant sections of the OIE manual.

(b)

Timing: the animals shall be tested twice: the first within two days from the date of their arrival in the quarantine station and the second at least 42 days from the date of the first test.

(c)

Options for action following testing: If any animal displays evidence of exposure to MCF, then none of the animals present in the quarantine station shall be considered as eligible for introduction into the Union.

(a)

Test to be used: AGID or blocking ELISA, in accordance with the protocols described in the OIE manual, latest version.

(b)

Timing: the animals shall be tested twice: the first within two days from the date of their arrival in the quarantine station and the second at least 42 days from the date of the first test.

(c)

Options for action following testing: animals tested positive to the test described in (a) shall be excluded from the group of animals in the quarantine facility and the other animals shall be re-tested starting at least 21 days from the date of the first positive test was performed: this shall 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 considered eligible for introduction into the Union.

‘BOV’

:

Model of veterinary certificate for fresh meat, including minced meat, of domestic bovine animals (including Bison and Bubalus species and their cross-breeds).

‘OVI’

:

Model of veterinary certificate for fresh meat, including minced meat, of domestic ovine animals (Ovis aries) and domestic caprine animals (Capra hircus).

‘POR’

:

Model of veterinary certificate for fresh meat, including minced meat, of domestic porcine animals (Sus scrofa).

‘EQU’

:

Model of veterinary certificate for fresh meat, excluding minced meat, of domestic solipeds (Equus caballus, Equus asinus and their cross-breeds).

‘RUF’

:

Model of veterinary certificate for fresh meat, excluding offal and minced meat, of farmed non-domestic animals of the order Artiodactyla (excluding bovine animals (including Bison and Bubalus species and their cross-breeds), Ovis aries, Capra hircus, Suidae and Tayassuidae), and of the families Rhinocerotidae and Elephantidae.

‘RUW’

:

Model of veterinary certificate for fresh meat, excluding offal and minced meat, of wild non-domestic animals of the order Artiodactyla (excluding bovine animals (including Bison and Bubalus species and their cross-breeds), Ovis aries, Capra hircus, Suidae and Tayassuidae), and of the families Rhinocerotidae and Elephantidae.

‘SUF’

:

Model of veterinary certificate for fresh meat, excluding offal and minced meat, of farmed non-domestic animals belonging to the Suidae, Tayassuidae, or Tapiridae families.

‘SUW’

:

Model of veterinary certificate for fresh meat, excluding offal and minced meat, of wild non-domestic animals belonging to the Suidae, Tayassuidae, or Tapiridae families.

‘EQW’

:

Model of veterinary certificate for fresh meat, excluding offal and minced meat, of wild solipeds belonging to the subgenus Hippotigris (zebra).

‘A’

:

guarantees regarding the maturation, pH measurement and boning of fresh meat, excluding offal, certified according to the models of veterinary certificates BOV (point II.2.6), OVI (point II.2.6), RUF (point II.2.7) and RUW (point II.2.4).

‘C’

:

guarantees regarding the laboratory test for classical-swine-fever in the carcases from which fresh meat was obtained, certified according to the model of veterinary certificate SUW (point II.2.3 B).

‘D’

:

guarantees regarding swill feed on holding(s) of animals from which fresh meat certified was obtained according to the model of veterinary certificate POR (point II.2.3 d).

‘E’

:

guarantees regarding tuberculosis test in the animals from where fresh meat certified was obtained, according to the model of veterinary certificate BOV (point II.2.4 d).

‘F’

:

guarantees regarding the maturation and de-boning of fresh meat, excluding offal, certified according to the models of veterinary certificates BOV (point II.2.6), OVI (point II.2.6), RUF (point II.2.6) and RUW (point II.2.7).

‘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 veterinary certificates RUF (point II.1.7) and RUW (point II.1.8).

‘H’

:

supplementary guarantees required for Brazil concerning animal contacts, vaccination programmes and surveillance. However as the State of Santa Catarina in Brazil does not vaccinate against foot and mouth disease, the reference to a vaccination programme is not applicable for meat coming from animals originating and slaughtered in that State.

*

Requirements as in accordance with the Agreement between the European Community and the Swiss Confederation on trade in agricultural products (OJ L 114, 30.4.2002, p. 132).

-

No certificates are laid down and fresh meat imports are prohibited (except for those species where indicated in the line comprising the entry for the whole country).

(a)

Veterinary certificates shall be issued by the exporting third country, based on the models set out in Part 2 of Annexes I, II and IV and Annex III according to the layout of the model that corresponds to the live animals/fresh meat 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 the Member State of destination imposes, for the live animals/fresh meat concerned, additional certification requirements, attestations to certify that those requirements are fulfilled shall also be incorporated in the original form of the veterinary certificate.

(b)

Where the model certificate states that certain statements shall be kept as appropriate, statements which are not relevant, may be crossed out and initialled and stamped by the certifying officer, or completely deleted from the certificate.

(c)

A separate and unique certificate must be provided for the live animals/fresh meat that are exported from a territory or territories of the same exporting country appearing in columns 2 and 3 of Part 1 of Annex I, II or IV which are consigned to the same destination and transported in the same railway wagon, lorry, aircraft or ship.

(d)

The original of each certificate shall consist of a single sheet of paper, or, where more text is required it must be in such a form that all sheets of paper required are part of an integrated whole and indivisible.

(e)

The veterinary certificate shall be drawn up in at least one of the official languages of the Member State of the border inspection post of introduction of the consignment into the Union and of the Member State of destination. However, those Member States may authorise the certificate to be drawn up in the official language of another Member State, and accompanied, if necessary, by an official translation.

(f)

If for reasons of identification of the items of the consignment (schedule in point I.28 of the model veterinary certificate), additional sheets of paper are attached to the certificate, those sheets of paper shall also be considered as forming part of the original of the certificate by the application of the signature and stamp of the certifying officer, on each of the pages.

(g)

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

(h)

The original of the certificate must be completed and signed by an official veterinarian or by another designated official inspector where this is provided for in the model veterinary certificate. In the case of live animals, the certificate must be completed and signed within 24 hours prior to loading of the consignment for introduction into the Union. The competent authorities of the exporting third country shall ensure that rules of certification equivalent to those laid down in Directive 96/93/EC (1) are followed.

The colour of the signature shall be different from that of the printing. This requirement also applies to stamps other than those embossed or watermarked.

(i)

The certificate reference number referred to in boxes I.2 and II.a. must be issued by the competent authority.