Regulation (EC) No 999/2001 of the European Parliament and of the CouncilShow full title

ANNEX X U.K. REFERENCE LABORATORIES, SAMPLING AND LABORATORY ANALYSIS METHODS

CHAPTER A U.K. National reference laboratories

1. The designated national reference laboratory is to: U.K.

(a)

have at its disposal facilities and expert personnel enabling it to show at all times, and especially when the disease in question first appears, the type and strain of the agent of TSE, and to confirm results obtained by regional diagnostic laboratories. Where it is not capable of identifying the strain-type of the agent, it shall set up a procedure to ensure that the identification of the strain is referred to the Community reference laboratory;

(b)

verify diagnostic methods used in regional diagnostic laboratories;

(c)

be responsible for coordination of diagnostic standards and methods within the Member State. To this end, it:

• may provide diagnostic reagents to laboratories approved by the Member State;

• is to control the quality of all diagnostic reagents used in the Member State;

• is to periodically arrange comparative tests;

• is to hold isolates of the agents of the disease in question, or corresponding tissues containing such agents, coming from cases confirmed in the Member State;

• is to ensure confirmation of results obtained in diagnostic laboratories designated by the Member State;

(d)

is to cooperate with the Community reference laboratory.

2. However, by way of derogation from point 1, Member States which do not have a national reference laboratory are to use the services of the Community reference laboratory or of national reference laboratories in other Member States. U.K.

[F1 3. The national reference laboratories are: U.K.

Austria:	Bundesanstalt für Tierseuchenbekämpfung, Mödling Robert Koch Gasse 17 A-2340 Mödling
Belgium:	CERVA-CODA-VAR Centre d'Étude et de Recherches Vétérinaires et Agrochimiques Centrum voor Onderzoek in Diergeneeskunde en Agrochemie Veterinary and Agrochemical Research Centre Groeselenberg 99 B-1180 Bruxelles
[F2 Cyprus:	Εργαστήριο Αναφοράς για τις Ασθένειες των Ζώων, Κτηνιατρικές Υπηρεσίες,1417 Λευκωσία (National Reference Laboratory for Animal Health Veterinary Services CY-1417 Nicosia)
Czech Republic:	Státní veterinární ústav Jihlava, Rantířovská 93, 586 05 Jihlava]
Denmark:	Danish Veterinary Laboratory Bülowsvej 27 DK-1790 Copenhagen V
[F2 Estonia:	Veterinaar- ja Toidulaboratoorium Kreutzwaldi 30 51006 Tartu]
Finland:	Eläinlääkintä- ja elintarvikelaitos Hämeentie 57 FIN-00550 Helsinki
France:	Agence Française de Sécurité Sanitaire des Aliments Laboratoire de pathologie bovine 31, avenue Tony Garnier BP 7033 F-69342 Lyon Cedex
Germany:	Bundesforschungsanstalt für Viruskrankheiten der Tiere Anstaltsteil Insel Riems Boddenblick 5A D-17498 Insel Riems
[F3 Greece:	Ministry of Agriculture Veterinary Laboratory of Larisa 7th km of Larisa — Trikala Highway GR-411 10Larisa (rapid tests and immunological tests) Laboratory of Gross Pathology Faculty of Veterinary Medicine Aristotelian University of Thessaloniki Giannitson & Voutyra St. GR-546 27 Thessaloniki (histopathology)]
[F2 Hungary:	Országos Állategészségügyi Intézet (OÁI) Pf. 2. Tábornok u. 2. HU-1581-Budapest]
Ireland:	Central Veterinary Research Laboratory Abbotstown Castleknock Dublin 15 Ireland
Italy:	Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d'Aosta CEA Via Bologna I-148-10150 Torino
[F2 Latvia:	Valsts veterinārmedicīnas diagnostikas centrs Lejupes iela 3 LV-1076 Rīga
Lithuania:	Nacionalinė veterinarijos laboratorija J. Kairiūkščio g. 10 LT-2021 Vilnius]
Luxembourg:	CERVA-CODA-VAR Centre d'Étude et de Recherches Vétérinaires et Agrochimiques Centrum voor Onderzoek in Diergeneeskunde en Agrochemie Veterinary and Agrochemical Research Centre Groeselenberg 99 B-1180 Bruxelles
[F2 Malta:	National Veterinary Laboratory Marsa Malta]
Netherlands:	Instituut voor Dierhouderij en Diergezondheid, ID-DLO Lelystad Edelhertweg 15 Postbus 658200 AB Lelystad Netherlands
[F2 Poland:	Laboratorium Zakładu Wirusologii Państwowego Instytutu Weterynaryjnego Al. Partyzantów 57 PL-24-100 Puławy]
Portugal:	Laboratório Nacional de Investigação Veterinária Estrada de Benfica, 701 P-1500 Lisboa
[F2 Slovakia:	Neuroimunologický ústav SAV Dúbravská cesta 9 SK-942 45 Bratislava
Slovenia:	Nacionalni veterinarski inštitut Gerbičeva 60 SI-1000 Ljubljana]
Spain:	Laboratorio de la Facultad de Veterinaria Departamento de Patología Animal (Anatomía Patológica) Zaragoza Spain (BSE and scrapie, methods other than rapid tests) Laboratorio Central de Veterinaria de Algete Madrid Spain (rapid tests) Centro de Investigacion en Sanidad Anímal (CISA) Crta, De Algete al Casar de Talamanca 28130 Valdeolmos (Madrid) Spain (TSEs other than BSE or scrapie)
Sweden:	National Veterinary Institute S-751 89 Uppsala
United Kingdom	Veterinary Laboratories Agency Woodham Lane New Haw Addlestone Surrey KT15 3NB United Kingdom]

Textual Amendments

F2 Inserted by Act concerning the conditions of accession of the Czech Republic, the Republic of Estonia, the Republic of Cyprus, the Republic of Latvia, the Republic of Lithuania, the Republic of Hungary, the Republic of Malta, the Republic of Poland, the Republic of Slovenia and the Slovak Republic and the adjustments to the Treaties on which the European Union is founded.

F3 Substituted by Commission Regulation (EC) No 1053/2003 of 19 June 2003 amending Regulation (EC) No 999/2001 of the European Parliament and of the Council as regards rapid tests (Text with EEA relevance).

Textual Amendments

F1 Substituted by Commission Regulation (EC) No 1248/2001 of 22 June 2001 amending Annexes III, X and XI to Regulation (EC) No 999/2001 of the European Parliament and of the Council as regards epidemio-surveillance and testing of transmissible spongiform encephalopathies.

CHAPTER B U.K. Community reference laboratory

1. The Community reference laboratory for TSEs is: U.K.

The Veterinary Laboratories Agency

Woodham Lane

New Haw

Addlestone

Surrey KT15 3NB

United Kingdom

2. The functions and duties of the Community reference laboratory are: U.K.

(a)

to coordinate, in consultation with the Commission, the methods employed in the Member States for diagnosing BSE, specifically by:

• storing and supplying corresponding tissues containing the agent, for the development or production of the relevant diagnostic tests or for typing strains of the agent;

• supplying standard sera and other reference reagents to the national reference laboratories in order to standardise the tests and reagents used in the Member States;

• building up and retaining a collection of corresponding tissues containing the agents and strains of TSEs;

• organising periodic comparative tests of diagnostic procedures at Community level;

• collecting and collating data and information on the methods of diagnosis used and the results of tests carried out in the Community;

• characterising isolates of the TSE agent by the most up-to-date methods to allow greater understanding of the epidemiology of the disease;

• keeping abreast of trends in surveillance, epidemiology and prevention of TSEs throughout the world;

• maintaining expertise on prion diseases to enable rapid differential diagnosis;

• acquiring a thorough knowledge of the preparation and use of diagnostic methods used to control and eradicate TSEs;

(b)

to assist actively in the diagnosis of outbreaks of TSEs in Member States by studying samples from TSE-infected animals sent for confirmatory diagnosis, characterisation and epidemiological studies;

(c)

to facilitate the training or retraining of experts in laboratory diagnosis with a view to the harmonisation of diagnostic techniques throughout the Community.

[F4 CHAPTER C U.K. Sampling and laboratory testing

1. Sampling U.K.

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 U.K.

Any laboratory examination for TSE shall be carried out in laboratories approved for that purpose by the competent authority.

3. Methods and protocols U.K.

3.1. Laboratory testing for the presence of BSE in bovine animals U.K.

(a) Suspect cases U.K.

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 U.K.

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 U.K.

(a) Suspect cases U.K.

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 U.K.

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 U.K.

(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 U.K.

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. U.K.

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.

[F5 4. Rapid tests U.K.

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 for the monitoring of BSE in bovine animals:

• immuno-blotting test based on a Western blotting procedure for the detection of the protease-resistant fragment PrP ^Res (Prionics-Check Western test),

• chemiluminescent ELISA test involving an extraction procedure and an ELISA technique, using an enhanced chemiluminescent reagent (Enfer test & Enfer TSE Kit version 2.0, automated sample preparation),

• sandwich immunoassay for PrP ^Res carried out following denaturation and concentration steps (Bio-Rad TeSeE test),

• microplate based immunoassay (ELISA) which detects protease resistant PrP ^Res 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 PrP ^Sc (some fraction of the protease resistant PrP ^Sc is equivalent to PrP ^Res ) and to PrP ^C (InPro CDI-5 test),

• chemiluminescent ELISA for qualitative determination of PrP ^Sc (CediTect BSE test),

• immunoassay using a chemical polymer for selective PrP ^Sc capture and a monoclonal detection antibody directed against conserved regions of the PrP molecule (IDEXX HerdChek BSE Antigen Test Kit, EIA),

• microplate based chemiluminiscent immunoassay for the detection of PrP ^Sc in bovine tissues (Institut Pourquier Speed’it BSE),

• lateral flow immunoassay using two different monoclonal antibodies to detect Proteinase K resistant PrP fractions (Prionics Check PrioSTRIP),

• two-sided immunoassay using two different monoclonal antibodies directed against two epitopes presented in a highly unfolded state of bovine PrP ^Sc (Roboscreen Beta Prion BSE EIA Test Kit),

• sandwich ELISA for the detection of Proteinase K (PK) resistant PrP ^Sc (Roche Applied Science PrionScreen).

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 for the monitoring of TSE in small ruminants:

• immuno-blotting test based on a Western blotting procedure for the detection of the protease-resistant fragment PrP ^Res (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 PrP ^Res 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 PrP ^Res 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 PrP ^Sc (some fraction of the protease resistant PrP ^Sc is equivalent to PrP ^Res ) and to PrP ^C (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.]

Textual Amendments

F5 Substituted by Commission Regulation (EC) No 260/2005 of 16 February 2005 amending Regulation (EC) No 999/2001 of the European Parliament and of the Council as regards rapid tests (Text with EEA relevance).

5. Alternative tests U.K.

(To be defined)]

Textual Amendments

F4 Substituted by Commission Regulation (EC) No 36/2005 of 12 January 2005 amending Annexes III and X to Regulation (EC) No 999/2001 of the European Parliament and of the Council as regards epidemio-surveillance for transmissible spongiform encephalopathies in bovine, ovine and caprine animals (Text with EEA relevance).