2.

In Annex X, Chapter C is replaced by the following: ‘CHAPTER C Sampling and laboratory testing 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 International Office for Epizooties (IOE/OIE) (“the Manual”). 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 Community Reference Laboratory. In particular the competent authority shall try to collect part of the cerebellum and the whole brain stem of small ruminants and shall keep at least half of the collected tissues fresh but not frozen until the result of the rapid or confirmatory test is negative. 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 laboratories approved 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 be subject to a histopathological examination as laid down in the latest edition of the Manual, except where the material is autolysed. Where the result of the histopathological examination is inconclusive or negative or where the material is autolysed, the tissues shall be subjected to an examination by one of the other diagnostic methods laid down in the Manual (immunocytochemistry, immuno-blotting or demonstration of characteristic fibrils by electron microscopy). However, rapid tests cannot be used for this purpose. If the result of one of those examinations is positive, the animals shall be regarded 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 (Monitoring in bovine animals) shall be examined by a rapid test. When the result of the rapid test is inconclusive or positive, the sample shall immediately be subject to confirmatory examinations in an official laboratory. The confirmatory examination shall start by a histopathological examination of the brainstem as laid down in the latest edition of the Manual, except where the material is autolysed or otherwise not suitable for examination by histopathology. Where the result of the histopathological examination is inconclusive or negative or where the material is autolysed, the sample shall be subjected to an examination by one of the other diagnostic methods referred to in (a). An animal shall be regarded a positive BSE case, if the result of the rapid test is positive or inconclusive, and either — the result of the subsequent histopathological examination is positive, or — the result of another diagnostic method referred to in (a) is positive. 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 be subject to a histopathological examination as laid down in the latest edition of the Manual, except where the material is autolysed. Where the result of the histopathological examination is inconclusive or negative or where the material is autolysed, the sample shall be subjected to an examination by immunocytochemistry, immuno-blotting or demonstration of characteristic fibrils by electron microscopy, as laid down in the Manual. However, rapid tests cannot be used for this purpose. If the result of one of those examinations is positive, the animal shall be regarded a positive scrapie case. (b) Scrapie 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. When the result of the rapid test is inconclusive or positive, the brainstem shall immediately be sent to an official laboratory for confirmatory examinations by immunocytochemistry, immuno-blotting or demonstration of characteristic fibrils by electron microscopy, as referred to in (a). If the result of the confirmatory examination is negative or inconclusive, additional confirmatory testing shall be carried out according the guidelines of the Community Reference Laboratory. If the result of one of the confirmatory examination is positive, the animal shall be regarded a positive scrapie case. (c) Further examination of positive scrapie cases (i) Primary molecular testing with a discriminatory immuno-blotting Samples from clinical suspect cases and from animals tested in accordance with Annex III, Chapter A, Part II, points 2 and 3 which are regarded as positive scrapie cases following the examinations referred to in points (a) or (b), or which display characteristics which are deemed by the testing laboratory to merit investigation, shall be forwarded for further examination by a primary molecular typing method to: — Agence Française de Sécurité Sanitaire des Aliments, Laboratoire de pathologie bovine, 31, avenue Tony Garnier, BP 7033, F-69342, Lyon Cedex, France, or — Veterinary Laboratories Agency, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom, or — to a laboratory, appointed by the competent authority, which has participated successfully in proficiency testing organised by the Community Reference Laboratory for the use of a molecular typing method, or — on a provisional basis until 1 May 2005, the laboratories approved for this purpose by the CRL panel of experts. (ii) Ring trial with additional molecular testing methods Samples from scrapie cases in which the presence of BSE cannot be excluded according to the guidelines issued by the Community Reference Laboratory by the primary molecular testing referred to in (i), shall be forwarded immediately to the laboratories listed in point (d) after consultation with the Community Reference Laboratory, and with all the relevant information available. They shall be submitted to a ring trial with at least: — a second discriminatory immuno-blotting, — a discriminatory immunocytochemistry, and — a discriminatory ELISA (Enzyme linked ImmunoSorbent Assay) carried out in the laboratories approved for the relevant method as listed in point (d). Where samples are unsuitable for immunocytochemistry, the Community Reference Laboratory will direct appropriate alternative testing within the ring trial. The results shall be interpreted by the Community Reference Laboratory assisted by a panel of experts including a representative of the relevant National Reference Laboratory. The Commission shall be informed immediately about the outcome of that interpretation. Samples indicative for BSE by three different methods and samples inconclusive in the ring trial shall be further analysed by a mouse bioassay for final confirmation. Further testing of samples taken from infected flocks on the same holding in accordance with the provisions of Annex III, Chapter A, Part II, point 5, shall be carried out in accordance with the advice of the Community Reference Laboratory, after consultation with the relevant National Reference Laboratory. (d) Laboratories approved for performing further examination by molecular typing methods The laboratories approved for further molecular typing are: Agence Française de Sécurité Sanitaire des Aliments Laboratoire de pathologie bovine 31, avenue Tony Garnier BP 7033 F-69342 Lyon Cedex Centre CEA Fontenay-aux-Roses, BP 6 F-92265 Fontenay-aux-Roses Cedex Service de Pharmacologie et d’Immunologie Centre CEA Saclay, bâtiment 136 F-91191 Gif-sur-Yvette Cedex Veterinary Laboratories Agency Woodham Lane New Haw Addlestone Surrey KT15 3NB United Kingdom 3.3. Laboratory testing for the presence of TSEs in species other than those referred to in points 3.1. and 3.2. Where methods and protocols are established for tests carried out to confirm the suspected presence of a TSE in a species other than bovine, ovine and caprine, they shall include at least a histopathological examination of brain tissue. The competent authority may also require laboratory tests such as immunocytochemistry, immuno-blotting, demonstration of characteristic fibrils by electron microscopy or other methods designed to detect the disease associated form of the prion protein. In any case at least one other laboratory examination shall be carried out if the initial histopathological examination is negative or inconclusive. At least three different examinations shall be carried out in the event of the first appearance of the disease. In particular, where BSE is suspected in a species other than bovine animals, samples shall be submitted for strain-typing, where possible. 4. Rapid tests For the purposes of carrying out the rapid tests in accordance with Article 5(3) and Article 6(1), the following methods shall be used as rapid tests: — immuno-blotting test based on a western blotting procedure for the detection of the protease-resistant fragment PrPRes (Prionics-Check Western test), — chemiluminescent ELISA test involving an extraction procedure and an ELISA technique, using an enhanced chemiluminescent reagent (Enfer test), — sandwich immunoassay for PrPRes carried out following denaturation and concentration steps (Bio-Rad TeSeE test, the former Bio-Rad Platelia test), — microplate based immunoassay (ELISA) which detects protease-resistant PrPRes with monoclonal antibodies (Prionics-Check LIA test), — automated conformation-dependent immunoassay comparing the reactivity of a detection antibody to the protease-sensitive and protease-resistant forms of PrPSc (some fraction of the protease-resistant PrPSc is equivalent to PrPRes) and to PrPC (InPro CDI-5 test). The producer of the rapid tests must have a quality assurance system in place agreed by the Community Reference Laboratory, which ensures that the test performance does not change. The producer must provide the test protocol to the Community Reference Laboratory. Modifications to rapid tests or to test protocols may only be made following advance notification to the Community Reference Laboratory, and provided that the Community Reference Laboratory finds that the modification does not reduce the sensitivity, specificity or reliability of the rapid test. That finding shall be communicated to the Commission and to the National Reference Laboratories. 5. Alternative tests (To be defined)’