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1. Sampling Any samples intended to be examined for the presence of a TSE shall be collected using the methods and protocols laid down in the latest edition of the Manual for diagnostic tests and vaccines for Terrestrial Animals of the World Organisation for Animal Health (OIE) (the Manual). In addition to, or in the absence of, OIE methods and protocols, and to ensure that sufficient material is available, the competent authority shall ensure the use of sampling methods and protocols in accordance with guidelines issued by the EU reference laboratory.

In particular the competent authority shall collect the appropriate tissues, according to the available scientific advice and the guidelines of the EU reference laboratory, in order to ensure the detection of all known strains of TSE in small ruminants and shall keep at least half of the collected tissues fresh but not frozen until the result of the rapid test is negative. Where the result is positive or inconclusive the residual tissues must be subject to confirmatory testing, and be processed subsequently in accordance with the EU reference laboratory guidelines on discriminatory testing and classification — ‘TSE strain characterisation in small ruminants: A technical handbook for National Reference Laboratories in the EU’. The samples shall be correctly marked as to the identity of the sampled animal.

2. Laboratories Any laboratory examination for TSE shall be carried out in official diagnostic laboratories designated for that purpose by the competent authority.

3. Methods and protocols 3.1. Laboratory testing for the presence of BSE in bovine animals (a) Suspect cases

Samples from bovine animals sent for laboratory testing pursuant to the provisions of Article 12(2) shall immediately be subjected to confirmatory examinations using at least one of the following methods and protocols laid down in the latest edition of the Manual:

(i) the immunohistochemical (IHC) method;

(ii) Western blot;

(iii) the demonstration of characteristic fibrils by electron microscopy;

(iv) histopathological examination;

(v) the combination of rapid tests as laid down in the third subparagraph. If the histopathological examination is inconclusive or negative, the tissues shall be submitted to a further examination by one of the other confirmatory methods and protocols. Rapid tests may be used for both primary screening of suspect cases and, if inconclusive or positive, for subsequent confirmation, according to the guidelines from the EU reference laboratory —‘OIE rules for the official confirmation of BSE in bovines (based on an initial reactive result in an approved rapid test) by using a second rapid test’, and provided that:

(i) the confirmation is carried out in a national reference laboratory for TSEs; and

(ii) one of the two rapid tests is a Western blot; and (iii) the second rapid test used:

— includes a negative tissue control and a bovine BSE sample as positive tissue control,

— is of a different type than the test used for the primary screening; and (iv) if a rapid Western blot is used as the first test, the result of that test must be documented and the blot image submitted to the national reference laboratory for TSEs; and (v) where the result of the primary screening is not confirmed by the subsequent rapid test, the sample must be subjected to an examination by one of the other confirmatory methods; where the histopathological examination is used for that purpose, but proves to be inconclusive or negative, the tissues must be submitted to a further examination by one of the other confirmatory methods and protocols. If the result of one of the confirmatory examinations referred to in points (i) to (v) of the first subparagraph is positive, the animal shall be regarded as a positive BSE case.

(b) BSE monitoring Samples from bovine animals sent for laboratory testing pursuant to the provisions of Annex III, Chapter A, Part I shall be examined by a rapid test.

When the result of the rapid test is inconclusive or positive, the sample shall immediately be subjected to confirmatory examinations using at least one of the following methods and protocols laid down in the latest edition of the Manual:

(i) the immunohistochemical (IHC) method;

(ii) Western blot;

(iii) the demonstration of characteristic fibrils by electron microscopy;

(iv) histopathological examination;

(v) the combination of rapid tests as laid down in the fourth subparagraph. Where the histopathological examination is inconclusive or negative, the tissues shall be submitted to a further examination by one of the other confirmatory methods and protocols. Rapid tests may be used for both primary screening and, if inconclusive or positive, for subsequent confirmation, according to the guidelines from the EU reference laboratory — ‘OIE rules for the official confirmation of BSE in bovines (based on an initial reactive result in an approved rapid test) by using a second rapid test, and provided that’:

(i) the confirmation is carried out in a national reference laboratory for TSEs; and (ii) one of the two rapid tests is a Western blot; and

(iii) the second rapid test used:

— includes a negative tissue control and a bovine BSE sample as positive tissue control,

— is of a different type than the test used for the primary screening; and (iv) if a rapid Western blot is used as the first test, the result of that test must be documented and the blot image submitted to the national reference laboratory for TSEs; and (v) where the result of the primary screening is not confirmed by the subsequent rapid test, the sample must be subjected to an examination by one of the other confirmatory methods; where the histopathological examination is used for that purpose, but proves to be inconclusive or negative, the tissues must be submitted to a further examination by one of the other confirmatory methods and protocols. An animal shall be regarded a positive BSE case if the result of the rapid test is inconclusive or positive, and at least one of the confirmatory examinations referred to in points (i) to (v) of the second subparagraph is positive.

(c) Further examination of positive BSE cases

Samples from all positive BSE cases shall be forwarded to a laboratory, appointed by the competent authority, which has participated successfully in the latest proficiency testing organised by the EU reference laboratory for discriminatory testing of confirmed BSE cases, where they shall be further tested in accordance with the methods and protocols laid down in the EU reference laboratory's method for the classification of bovine TSE isolates (a two-blot method for the provisional classification of bovine TSE isolates).

3.2. Laboratory testing for the presence of TSE in ovine and caprine animals (a) Suspect cases Samples from ovine and caprine animals sent for laboratory testing pursuant to the provisions of Article 12(2) shall immediately be subjected to confirmatory examinations using at least one of the following methods and protocols laid down in the latest edition of the Manual:

(i) the immunohistochemical (IHC) method;

(ii) Western blot;

(iii) the demonstration of characteristic fibrils by electron microscopy;

(iv) histopathological examination.

In case the histopathological examination is inconclusive or negative, the tissues shall be submitted to a further examination by one of the other confirmatory methods and protocols. Rapid tests may be used for primary screening of suspect cases. Such tests may not be used for subsequent confirmation. Where the result of the rapid test used for primary screening of suspect cases is positive or inconclusive, the sample shall be subjected to an examination by one of the confirmatory examinations referred to in points (i) to (iv) of the first subparagraph. Where the histopathological examination is used for that purpose, but proves to be inconclusive or negative, the tissues shall be submitted to a further examination by one of the other confirmatory methods and protocols. If the result of one of the confirmatory examinations referred to in points (i) to (iv) of the first subparagraph is positive, the animal shall be regarded as a positive TSE case.

(b) TSE monitoring

Samples from ovine and caprine animals sent for laboratory testing pursuant to the provisions of Annex III, Chapter A, Part II (Monitoring in ovine and caprine animals) shall be examined by a rapid test, in order to ensure the detection of all known strains of TSE. When the result of the rapid test is inconclusive or positive, the sampled tissues shall immediately be sent to an official laboratory for confirmatory examinations by histopathology, immunohistochemistry, Western blotting or demonstration of characteristic fibrils by electron microscopy, as referred to in point (a). If the result of the confirmatory examination is negative or inconclusive, the tissues shall be submitted to a further examination by immunohistochemistry or Western blotting. If the result of one of the confirmatory examinations is positive, the animal shall be regarded as a positive TSE case.

(c) Further examination of positive TSE cases

Samples that, following the examinations referred to in points (a) or (b), are regarded as positive TSE cases, but which are not considered atypical cases, shall be examined to exclude the presence of BSE only when they come from an index case. Other cases, which display characteristics that, according to the testing laboratory, merit investigation, shall also be examined to exclude the presence of BSE.

(i) Primary molecular testing with a discriminatory Western blotting method For the exclusion of the presence of BSE, samples shall be examined by a discriminatory Western blotting method, listed in the guidelines of the EU reference laboratory. The discriminatory examination shall be performed by an official laboratory, designated by the competent authority, which has participated successfully in the latest proficiency testing organised by the EU reference laboratory for the use of such a method.

(ii) Secondary molecular testing with additional molecular testing methods