(a)  herd means an animal or group of animals kept on a holding (within the meaning of Article 2 (b) of Directive 92/102/EEC) as an epidemiological unit; if more than one herd is kept on a holding, each of these herds shall form a distinct unit and shall have the same health status;

(b)  animal for slaughter means a bovine animal (including the species Bison bison and Bubalus bubalus) or swine intended to be taken to a slaughterhouse or assembly centre from which it may only move to slaughter;

(c)  animals for breeding or production means bovine animals (including the species Bison bison and Bubalus bubalus) and swine other than those referred to in (b), including those intended for breeding, milk or meat production, or draft purposes, shows or exhibition with the exception of animals taking part in cultural and sporting events;

(d)  officially tuberculosis-free bovine herd means a bovine herd which satisfies the conditions laid down in ►  Annex A.I, paragraphs 1 and 2 ◄ ;

(e)  officially tuberculosis-free Member State or region of a Member State means a Member State or part of a Member State which satisfies the conditions laid down in ►  Annex A.I, paragraphs 4 and 5 ◄ ;

(f)  officially brucellosis-free bovine herd means a bovine herd which satisfies the conditions laid down in ►  Annex A.II, paragraphs 1 and 2 ◄ ;

(g)  officially brucellosis-free region means a region of a Member State which satisfies the conditions laid down in Annex A.II, paragraphs 7, 8 and 9;

(h)  officially brucellosis-free Member State means a Member State which satisfies the conditions laid down in ►  Annex A.II, paragraphs 7, 8 and 9 ◄ ;

(i)  brucellosis-free bovine herd means a bovine herd which satisfies the conditions laid down in ►  Annex A.II, paragraphs 4 and 5 ◄ ;

(j)  officially enzootic-bovine-leukosis-free herd means a herd which satisfies the conditions laid down in Annex D, Chapter I, Sections A and B;

(k)  officially enzootic-bovine-leukosis-free Member State or region means a region or Member State which meets the requirements laid down in ►  Annex D, Chapter I, Sections E and F ◄ ;

(l)  official veterinarian means the veterinarian appointed by the competent authority of the Member State;

(m)  approved veterinarian means any veterinarian approved by the competent authority in accordance with the provisions of Article 14 (3) (B);

(n)  compulsorily notifiable diseases means the diseases listed in Annex E (I);

(o)  assembly centre means holdings, collection centres and markets, at which bovine animals or swine originating from different holdings are grouped together to form consignments of animals intended for trade. These assembly centres must be approved for trading purposes and meet the requirements laid down in Article 11;

(p)  region means that part of a Member State's territory which is at least 2 000 km2 in area and which is subject to inspection by the competent authorities and includes at least one of the following administrative regions:

(q)  dealer means any natural or legal person who buys and sells animals commercially either directly or indirectly, who has a regular turnover of these animals and who within a maximum of 30 days of purchasing animals resells them or relocates them from the first premises to other premises not within his ownership and meets the conditions laid down in Article 13.

(a) be subjected:

(b) not have been obtained from a holding or an area which for health reasons is subject to prohibition or restriction affecting the species involved in accordance with Community and/or national legislation;

(c) be identified as provided for in Directive 92/102/EEC;

(d) be identified in accordance with the provisions of Directive 92/102/EEC, in the case of swine and in accordance with the provisions of Regulation (EC) No 1760/2000 in the case of bovine animals;

(e) comply with the provisions of Articles 4 and 5.

(a) animals coming from approved assembly centres, such certification shall be:

(b) animals coming from an approved holding which is participating in the surveillance network provided for in Article 14, such certification shall be:

— have remained in a single holding of origin for a period of 30 days prior to loading, or since birth in the holding of origin where the animals are less than 30 days old. The official veterinarian must, on the basis of the official identification provided for in Article 3 (2) (c) and official records, be satisfied that the animals have complied with this condition and furthermore that the animals have originated in the Community or have been imported from a third country in compliance with Community animal health legislation.

— However, in the case of animals transiting through an approved assembly centre in the Member State of origin, the period during which the assembly of these animals takes place outside the holding of origin shall not exceed six days,

— with regard to animals imported from a third country into a Member State which is not that of ultimate destination, be transported to the Member State of destination as quickly as practicable under cover of a certificate issued under Article 7 of Directive 91/496/EEC,

— with regard to animals imported from a third country upon arrival at destination and before any further movements satisfy the requirements of this Directive, and in particular the residency requirement in the first indent, and may not be brought into the herd until the veterinarian responsible for that holding has ascertained that the animals in question are not likely to jeopardize the health status of the holding.

— If an animal from a third country is introduced into a holding no animal from the holding may be traded for 30 days following introduction unless the imported animal is isolated from all other animals on the holding.

(a)  ►M55  come from an officially tuberculosis-free bovine herd, and in the case of animals more than six weeks old, have reacted negatively to an intradermal tuberculin test carried out in accordance with the provisions of point 2.2 of Annex B either during the 30 days prior to leaving the herd of origin or in a place and under conditions to be defined in accordance with the procedure referred to in Article 17. ◄

This intradermal tuberculin test is not required if the animals originate in a Member State or part of a Member State recognized as officially tuberculosis free or in a Member State or part of a Member State with an approved surveillance network;

(b) in the case of uncastrated animals which come from an officially brucellosis-free bovine herd and more than 12 months old, have shown a brucella count lower that 30 international units (IU) of agglutination per millilitre when given a serum agglutination test (or any test approved by Standing Veterinary Committee (SVC) procedure following the adoption of the relevant protocols) carried out during the 30 days prior the leaving the herd of origin and complying with the provisions of Annex C Section A.

This serum agglutination test (or any test approved by SVC procedure following the adoption of the relevant protocols) is not required if the animals originate in a Member State or part of a Member State recognized as officially brucellosis free or in a Member State or part of a Member State with an approved surveillance network;

(c) come from an officially enzootic-bovine-leukosis-free herd and, if more than 12 months old, have reacted negatively to an individual test carried out during the 30 days prior to leaving the herd of origin and complying with the provisions of Annex D.

The test is not required if the animals originate in a Member State or part of a Member State recognized as officially enzootic-bovine-leukosis free or in a Member State or part of a Member State with an approved surveillance network;

(d) at no time between leaving the holding of origin and arriving at destination come into contact with bovine animals which meet only the requirements in paragraph 3;

(e) until 31 December 2000, not be subject to the test requirements laid down in (a) or (b) in the case of bovine animals aged less than 30 months intended for meat production which:

and provided that:

— have in the 30 days prior to embarkation undergone the appropriate tests laid down in Annexes B, C and D, with negative results,

— are taken on arrival in the country of destination directly to a slaughterhouse and are slaughtered there as soon as possible but at least within 72 hours of arrival, in accordance with animal health requirements.

— to a slaughterhouse, must be slaughtered there as soon as possible but at least within 72 hours of arrival, in accordance with animal health requirements, or

— to an approved assembly centre, must be removed after the market directly to a slaughterhouse to be slaughtered as soon as possible but at the latest within three working days of arrival at the assembly centre, in accordance with animal health requirements. At no time, between their arrival at the assembly centre and their arrival at the slaughterhouse, may they come into contact with cloven-hoofed animals other than animals that fulfil the conditions laid down in this Directive.

— the distribution of the disease in the Member State,

— the reasons for the programme, taking into consideration the importance of the disease and the programme's likely benefit in relation to its cost,

— the geographical area in which the programme will be implemented,

— the status categories to be applied to the animal establishments, the standards which must be attained in each category, and the test procedures to be used,

— the programme monitoring procedures, the results of which must be supplied at least annually to the Commission,

— the action to be taken if, for any reason, an establishment loses its status,

— the measures to be taken if the results of the tests carried out in accordance with the provisions of the programme are positive.

— the nature of the disease and the history of its occurrence in its territory,

— the results of surveillance testing based on serological, microbiological, pathological or epidemiological investigation and on the fact that the disease must by law be notified to the competent authorities,

— the period over which the surveillance was carried out,

— where applicable, the period during which vaccination against the disease has been prohibited and the geographical area concerned by the prohibition,

— the arrangements for verifying the absence of the disease.

(a) be under the control of an official veterinarian who shall ensure that, in particular, the provisions of Article 4 (1) and (2) are complied with;

(b) be located in an area which is not subject to prohibiiton or restrictions in accordance with relevant Community legislation and/or national legislation;

(c) be cleaned and disinfected before use, as required by the official veterinarian;

(d) they must have, taking into account the animal capacity of the assembly centre:

(e) admit only animals that are identified and come from herds that are officially free of tuberculosis, brucellosis and leukosis or slaughter animals meeting the conditions set out in the present Directive and in particular Article 6 (3). To this end, when animals are admitted the owner or person in charge of the centre shall ensure they are properly identified and accompanied by health documents or appropriate certificates for the species and categories involved;

(ee) comply with the provisions of Directive 98/58/EC and Regulation (EC) No 1/2005 ( 13 ) applicable to them;

(f) be regularly inspected in order to ascertain that the requirements for approval continue to be fulfilled.

— the name of the owner, the origin, date of entry and exit, number and identification of the bovine animals or the registration number of the holding of origin or of the herd of origin of the pigs entering the centre and their proposed destination,

— the registration number of the transporter and the licence number of the lorry delivering or collecting animals from the centre.

(a) for the carriage of animals they must use means of transport that are:

(b) they must either:

(a) places, dates and times of pick-up, and the name or business name and address of the holding or assembly centre where the animals are picked up;

(b) places, dates and times of delivery, and the name or business name and address of the consignee(s);

(c) the species and number of animals carried;

(d) date and place of disinfection;

(e) details of accompanying documentation including the number;

(f) expected duration of each journey.

(a) they must deal only in animals that are identified and come from herds that are officially free of tuberculosis, brucellosis and leukosis or slaughter animals meeting the conditions set out in this Directive and in particular Article 6 (3). To this end, the dealer shall ensure that the animals are properly identified and are accompanied by health documents as appropriate for the species involved.

However, the competent authority may authorize the marketing of identified animals which do not fulfil the conditions laid down in the first paragraph, in so far as they are brought direct to a slaughterhouse in the Member State of origin without passing through their facilities, for slaughter as soon as possible in order to prevent the spreading of diseases. The necessary provisions should be taken to ensure that such animals, when they reach the abattoir, cannot come into contact with other animals and that they are slaughtered apart from other animals;

(b) the dealer shall be required, either on the basis of the document accompanying the animals, or on the basis of identification numbers or marks on the animals, to keep a record or data base and to store the following data for at least three years:

(c) when the dealer keeps animals on his premises he shall ensure that:

(a) they must be under the control of an official veterinarian;

(b) they must be located in an area which is not subject to prohibition or restrictions in accordance with the relevant Community legislation or national legislation;

(c) they must have:

(d) be cleaned and disinfected before use, as required by the official veterinarian.

— the herds,

— the owner or any other natural or legal person responsible for the holding,

— the approved veterinarian or the official veterinarian responsible for the holding,

— the official veterinary service of the Member State,

— the official veterinary diagnostic laboratories or any other laboratory approved by the competent authority,

— a computer database.

A. Every owner of or person responsible for a holding must:

B. The approved veterinarians as provided for by Article 2 (2) (m) shall be under the control of the competent authority and must comply with the following requirements.

They must:

C. The computer database must contain at least the following information:

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(a) arrange for the journey to be completed or the animals returned to their place of departure by the most direct route, providing this course of action would not further jeopardize the health or welfare of the animals;

(b) arrange for the animals to be held in suitable accommodation with appropriate care in the event of interruption of the journey;

(c) arrange for the slaughter of the animals. The destination and use of such animals after slaughter shall be regulated:

(a) for bovine animals, from 31 December 1999;

(b) for a register of holdings of porcine animals, complying with the provisions laid down in Article 14(3)(c)(2) from 31 December 2000;

(c) for movements of porcine animals, complying with the provisions laid down in Article 14(3)(c)(3):

1. A bovine herd is officially tuberculosis-free if:

2. A bovine herd will retain officially tuberculosis-free status if:

3A. The officially tuberculosis-free status of a herd is to be suspended if:

3B. The officially tuberculosis-free status of the herd is to be withdrawn if the presence of tuberculosis is confirmed by the isolation of M. bovis on laboratory examination.

The competent authority may withdraw status if:

Tracing and checking is to be undertaken by the competent authority of any herd considered to be epidemiologically related. The officially tuberculosis-free status of a herd is to remain withdrawn until cleansing and disinfection of the premises and utensils has been completed and all animals over six weeks of age have reacted negatively to at least two consecutive tuberculin tests, the first no less than 60 days and the second no less than four months and no more than 12 months after the removal of the last positive reactor.

4. On the basis of information supplied in accordance with Article 8, a Member State or part of a Member State may be declared officially tuberculosis-free according to the procedure laid down in Article 17 if it meets the following conditions:

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▼M44

5. The Member State or part of a Member State will retain officially tuberculosis-free status if the conditions 4(a) to (d) continue to be met. However, if there is evidence of a significant change in the situation as regards tuberculosis in a Member State or part of a Member State which has been recognised as officially tuberculosis-free, the Commission may, in accordance with the procedure laid down in Article 17, take a Decision suspending or revoking the status until the requirements of the Decision have been fulfilled.

1. A bovine herd is officially brucellosis-free if:

2. A bovine herd will retain officially brucellosis-free status if:

3A. The officially brucellosis-free status of a herd is to be suspended if:

Where the animal has been slaughtered and is no longer available for testing, the suspension may be lifted if two serum agglutination tests, carried out in accordance with Annex C on all bovine animals in the herd over 12 months old, show a titre lower than 30 IU of agglutination per ml. The first test shall be carried out at least 30 days after the removal of the animal and the second at least 60 days later.

Where the animal has been isolated from the animals in the herd, it may be reintroduced into the herd and the status of the herd may be restored following:

3B. The officially brucellosis-free status of the herd is to be withdrawn if, as a result of laboratory tests or epidemiological investigations, brucella infection has been confirmed in the herd.

The status of the herd is not to be restored until either all bovine animals present in the herd at the time of the outbreak have been slaughtered, or the herd has been subject to check testing and all animals over 12 months of age have given negative results to two consecutive tests at 60-day intervals, the first being carried out not less than 30 days after removal of the positive animal(s).

In the case of bovine animals which were pregnant at the time of the outbreak, the final check must be carried out at least 21 days after the last animal pregnant at the time of the outbreak has calved.

4. A bovine herd is brucellosis-free if it complies with the conditions in 1(b) and (c) and when vaccination has been carried out as follows:

5. A bovine herd will retain brucellosis-free status if:

6A. The brucellosis-free status of a herd is to be suspended if:

Where the animal has been isolated, it may be reintroduced into the herd and the status of the herd may be restored, if it subsequently shows a serum agglutination titre lower than 30 IU of agglutination per ml and has given a negative result to a complement fixation test, or other test approved under the procedure set out in Article 17.

Where the animals have been slaughtered and are no longer available for testing, the suspension may be lifted if two serum agglutination tests, carried out in accordance with Annex C on all bovine animals in the holding over 12 months old, show a titre lower than 30 IU of agglutination per ml. The first test is to be carried out at least 30 days after the removal of the animal and the second at least 60 days later.

If the animals to be tested in the previous two subparagraphs are under 30 months old and have been vaccinated with live strain 19 vaccine they may be considered to be negative if they give a serum agglutination test result greater than 30 IU but less than 80 IU of agglutination per millilitre provided that, on the complement fixation test, they give a result less than 30 EEC units in the case of females vaccinated less than 12 months previously or less than 20 EEC units in all other cases.

6B. The brucellosis-free status of the herd is to be withdrawn if, as a result of laboratory tests of epidemiological investigations, brucella infection has been confirmed in a herd. The status of the herd is not to be restored until either all the bovine animals present in the herd at the time of the outbreak have been slaughtered or the herd has been subject to check testing and all unvaccinated animals over 12 months of age have given negative results to two consecutive tests at 60 day intervals, the first being carried out not less than 30 days after removal of the positive animal(s).

If all the animals to be tested referred to in the preceding paragraph are less than 30 months old and have been vaccinated with live strain 19 vaccine, they may be considered negative if they show a brucella titre of more than 30 IU but less than 80 IU of agglutination per ml, provided that in the complement fixation test they show a titre of less than 30 EEC units in the case of females vaccinated less than 12 months previously or a titre of less than 20 EEC units in all other cases.

In the case of bovine animals which were pregnant at the time of the outbreak, the final check must have been carried out at least 21 days after the last animal pregnant at the time of the outbreak has calved.

7. A Member State or a region of a Member State may be declared officially brucellosis-free according to the procedure laid down in Article 17 if it meets the following conditions:

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8. Subject to paragraph 9, a Member State or a region of a Member State declared officially brucellosis-free is to retain this status if:

9. A Member State or a region of a Member State declared officially brucellosis-free is to report the occurrence of all cases of brucellosis to the Commission. If there is evidence of a significant change in the situation as regards brucellosis in a Member State or part of a Member State which has been recognised as officially brucellosis-free, the Commission may according to the procedure laid down in Article 17 propose that the status be suspended or revoked until the requirements of the Decision have been fulfilled.

10. For the purposes of section II, a serological test means either a serum agglutination test, buffered brucella antigen test, complement fixation test, plasma agglutination test, plasma ring test, micro-agglutination test or individual blood ELISA, as described in Annex C. Any other diagnostic test approved under the procedure laid down in Article 17 and described in Annex C will also be accepted for the purposes of section II. A milk test means a milk ring test or a milk ELISA in accordance with Annex C.

2.1.4.1. pH: The pH is 6.5 to 7.5.

2.1.4.2. Phenol: If the preparation to be examined contains phenol, its concentration is not more than 5 g/l.

2.1.4.3. Sensitising effect: Use a group of three guinea-pigs that have not been treated with any material which will interfere with the test. On 3 occasions at intervals of five days inject intradermally into each guinea-pig a dose of the preparation to be examined equivalent to 500 IU in 0,1 ml. 15 to 21 days after the third injection inject the same dose (500 IU) intradermally into these animals and into a control group of three guinea-pigs of the same mass and which have not previously received injections of tuberculin. 24 to 28 hours after the last injections, the reactions of the two groups are not significantly different.

2.1.4.4. Toxicity: Use two guinea-pigs, each weighing not less than 250 g and which have not previously been treated with any material which will interfere with the test. Inject subcutaneously into each guinea-pig 0,5 ml of the preparation to be examined. Observe the animals for seven days. No abnormal effects occur during the observation period.

2.1.4.5. Sterility: It complies with the test for sterility prescribed in the monograph on Vaccines for veterinary use 4th Edition 2002 of the European Pharmacopoeia.

— the potency in International Units per millilitre,

— the name and quantity of any added substance,

— for freeze-dried preparations:

2.2.1. The following shall be recognised as official intradermal tuberculin tests:

2.2.2. The dose of tuberculin injected shall be:

2.2.3. The volume of each injection dose shall not exceed 0,2 ml.

2.2.4. Tuberculin tests shall be carried out by injecting tuberculin(s) into the skin of the neck. The injection sites shall be situated at the border of the anterior and middle thirds of the neck. When both avian and bovine tuberculins are injected in the same animal, the site for injection of avian tuberculins shall be about 10 cm from the crest of the neck and the site for the injection of bovine tuberculin about 12,5 cm lower on a line roughly parallel with the line of the shoulder or on different sides of the neck; in young animals in which there is not room to separate the sites sufficiently on one side of the neck, one injection shall be made on each side of the neck at identical sites in the centre of the middle third of the neck.

2.2.5. The technique of tuberculin testing and interpretation of reactions shall be as follows:

2.2.5.1.  Technique:

Injection sites shall be clipped and cleansed. A fold of skin within each clipped area shall be taken between the forefinger and thumb and measured with callipers and recorded. The dose of tuberculin shall then be injected by a method that ensures that the tuberculin is delivered intradermically. A short sterile needle, bevel edge outwards, with graduated syringe charged with tuberculin, inserted obliquely into the deeper layers of the skin may be used. A correct injection shall be confirmed by palpating a small pea-like swelling at each site of injection. The skin-fold thickness of each injection site shall be remeasured 72 hours (± 4 hours) after injection and recorded.

2.2.5.2.  Interpretation of reactions

The interpretation of reactions shall be based on clinical observations and the recorded increase(s) in skin-fold thickness at the sites of injection 72 hours after injection of tuberculin(s).

2.2.5.3. The interpretation of official intradermal tuberculin tests shall be as follows:

2.2.5.3.1. Single intradermal test:

Animals inconclusive to the single intradermal test shall be subjected to another test after a minimum of 42 days.

Animals which are not negative to this second test shall be deemed to be positive to the test.

Animals positive to the single intradermal test may be subjected to an intradermal comparative test if false positive reaction or interference reaction is suspected.

2.2.5.3.2. Intradermal comparative test for the establishment and maintenance of officially tuberculosis-free herd status:

Animals inconclusive to the intradermal comparative test shall be subjected to another test after a minimum of 42 days. Animals, which are not negative to this second test, shall be deemed to be positive to the test.

2.2.5.3.3. Officially tuberculosis-free herd status may be suspended and animals from the herd shall not be allowed to enter intra-Community trade until such time as the status of the following animals is resolved:

2.2.5.3.4. Where animals are required by Community legislation to be subjected to an intradermal test prior to movement, the test shall be interpreted so that no animal which shows an increase in skin-fold thickness greater than 2 mm or the presence of clinical signs is entered into intra-Community trade.

2.2.5.3.5. To enable detection of the maximum number of infected and diseased animals in a herd or in a region, Member States may modify the criteria for the interpretation of the test in order to achieve improved test sensitivity considering all inconclusive reactions referred in 2.2.5.3.1(b) and 2.2.5.3.2(b) as positive reactions.

(a) The brucellosis skin test shall not be used for the purpose of certification for intra-Community trade.

(b) The brucellosis skin test is one of the most specific tests for the detection of brucellosis in unvaccinated animals; however diagnosis must not be made on the basis of positive intradermal reactions alone.

(c) Bovine animals, tested with negative result in one of the serological tests defined in this Annex and reacting positively to the BST shall be regarded as infected or suspected to be infected.

(d) Bovine animals, tested with positive result in one of the serological tests defined in this Annex may be subject to a BST in order to support the interpretation of the serological test results; in particular where in officially brucellosis-free or brucellosis-free bovine herds a cross-reaction with antibodies against other bacteria cannot be excluded.

(a) the approval of the results of the validation studies demonstrating the reliability of the test method used in the Member State;

(b) determination of the maximum number of samples to be pooled in ELISA kits used;

(c) calibration of the standard secondary reference national standard sera (‘working standards’) against the primary international standard serum referred to in paragraph 2.1;

(d) quality checks of all antigens and ELISA kits batches used in the Member State;

(e) cooperation within the European Union Network of National Reference Laboratories for Brucellosis.

A. Officially enzootic-bovine-leukosis-free herd means a herd in which:

B. A herd shall retain officially enzootic-bovine-leukosis-free status provided:

C. The officially leukosis-free status of a herd is to be suspended if the conditions detailed in B are not fulfilled, or where as a result of laboratory tests or on clinical grounds one or more bovine animals are suspected of having enzootic bovine leukosis and the suspect animal(s) are immediately slaughtered.

D. The status is to remain suspended until the following requirements are complied with:

E. In accordance with the procedure in Article 17 and on the basis of information supplied in accordance with Article 8, a Member State or part of a Member State may be considered officially enzootic-bovine-leukosis-free if:

F. A Member State or a region of a Member State is to retain officially enzootic-bovine-leukosis-free status if:

G. The officially enzootic-bovine-leukosis-free status of a Member State or part of a Member State is to be suspended, in accordance with the procedure in Article 17 if, as a result of investigations carried out in accordance with paragraph F above, there is evidence of a significant change in the situation as regards enzootic bovine leukosis in a Member State or part of a Member State which has been recognised as officially enzootic-bovine-leukosis-free.

The officially enzootic-bovine-leukosis-free status may be restored in accordance with the procedure in Article 17 when the criteria laid down by the same procedure are fulfilled.

1. The antigen to be used in the test shall contain bovine leukosis virus glycoprotein. The antigen shall be standardised against the E05 serum.

2. The State institutes, national reference laboratories or official institutes designated in accordance with Article 6a for coordinating standards and methods of diagnosis of the tests for enzootic bovine leukosis shall be made responsible for calibrating the standard working antigen of the laboratory against the E05 serum.

3. The standard antigens used in the laboratory shall be submitted at least once a year to the State institutes, national reference laboratories or official institutes designated in accordance with Article 6a, for testing against the E05 serum. Apart from such standardisation, the antigen in use may be calibrated in accordance with the method described in Section B.

4. The reagents of the tests shall consist of:

5. A test pattern of seven moisture-free wells shall be cut in the agar to the bottom of the plate; the pattern shall consist of one central well and six wells in a circle around it.

Diameter of central well: 4 mm

Diameter of peripheral wells: 6 mm

Distance between central and peripheral wells: 3 mm

6. The central well shall be filled with the standard antigen. Peripheral wells 1 and 4 described in B.3 are filled with the known positive serum; wells 2, 3, 5 and 6 with the test sera. The wells shall be filled until the meniscus disappears.

7. This results in the following quantities being obtained:

8. Incubation shall be for 72 hours at room temperature (20 to 27 °C) in a closed humid chamber.

9. The test may be read at 24 and 48 hours but a final result shall not be obtained before 72 hours:

In inconclusive reactions the test may be repeated and concentrated serum utilised.

10. Any other well configuration or pattern may be utilised provided that the E05 serum diluted 1:10 in negative serum can be detected as positive.

1. Solutions and materials required:

2. Procedure:

Dissolve the agarose (1,6 %) in the Tris/HCl buffer by carefully heating to 100 °C. Place in 56 °C water bath for approximately 1 hour. Also, place the bovine leukosis serum dilutions in a 56 °C water bath.

Now mix 15 ml of the 56 °C agarose solution with the 15 ml bovine leukosis serum (1:10), quickly shake and pour 15 ml into each of two petri dishes. Repeat this procedure with the bovine leukosis serum diluted 1:5.

When the agarose has hardened, holes shall be made in it as follows:

3. Addition of antigen:

4. Additional instructions:

1. The material and reagents to be used shall be as follows:

2. Standardisation and sensitivity of test

The sensitivity of the ELISA shall be of such a level that the E05 serum is scored positive when diluted 10 times (serum samples) or 250 times (milk samples) more than the dilution obtained of individual samples when these are included in pools. In assays where samples (serum and milk) are tested individually, the E05 serum diluted 1 to 10 (in negative serum) or 1 to 250 (in negative milk) shall be scored positive when tested in the same assay dilution as used for the individual test samples. The institutes referred to in point 2 of Section A shall be responsible for checking the quality of the ELISA, and in particular for determining, for each production batch, the number of samples to be pooled on the basis of the count obtained for the E05 serum.

3. Conditions for use of the ELISA for enzootic bovine leukosis

— Foot-and-mouth disease

— Rabies

— Tuberculosis

— Brucellosis

— Contagious bovine pleuropneumonia

— Enzootic bovine leukosis

— Anthrax

— Rabies

— Brucellosis

— Classical swine fever

— African swine fever

— Foot-and-mouth disease

— Swine vesicular disease

— Anthrax

— Aujeszky's disease

— Infectious bovine rhinotracheitis

— Brucella suis infection

— Transmissible gastro-enteritis