1. SEGURIDAD ALIMENTARIA Y DERECHO ALIMENTARIO WORKSHOP ON BSE CONTROL MEASURES- KIEV MICROSCOPY OF ANIMAL COMPONENTS IN FEEDINGSTUFFS COMMISION DIRECTIVE 2003/126/EC Dr. Pedro Diaz Peralta PhDVM LLM Apr. 2011

2. WORKSHOP ON BSE CONTROL MEASURES- KIEV MICROSCOPY OF ANIMAL COMPONENTS The ban of use in animal feeding of processed animal proteins (PAPs) for farmed animals became in a effective reduction of the number of bovine spongiform encephalopathy cases. The European Commission established an official method to detect the presence of animal-derived constituents in feedstuffs, using microscopic examination. (Directive 2003/126/EC) This method allows easy identification of bone fragments among other animal constituents. The method was updated in 2009 by Regulation (EC) nº 152/2009, HOWEVER…

3. WORKSHOP ON BSE CONTROL MEASURES- KIEV TSE ROAD MAP- 2 European Commision Review on implementation of TSE measures: (Strategic Paper for TSE 2010- 2015) (Findings:) The Community Reference Laboratory for animal proteins (CRL- AP) has evaluated the strength of microscopic methods regarding the quantitative determination of animal constituents in feedingstuffs (as regards the possible introduction of tolerance limits) The preliminary conclussions revealed that the current method is not reliable for the purpose of cuantification MICROSCOPY OF ANIMAL COMPONENTS

4. WORKSHOP ON BSE CONTROL MEASURES- KIEV TSE ROAD MAP- 2 European Commision Review on implementation of TSE measures: (Strategic Paper for TSE 2010- 2015) (Findings:) In addition, the Community Reference Laboratory for animal proteins (CRL- AP) has evaluated other alternative methods with purposes of estimate PAP or Meat Bone Meal (MBM) which may identify the species (ruminants, pig, poultry) The mandatory treatment in rendering process (133 º C/ 3 bars, during 20 minutes) results in very small fragments of animal proteins which are difficult to detect by current detection methods MICROSCOPY OF ANIMAL COMPONENTS

5. TSE ROAD MAP- 2: Current situation Feed ban A: Authorized NA: Non authorized 4 With the exception of fishmeal in meal replacers for young bovines

6. WORKSHOP ON BSE CONTROL MEASURES- KIEV TSE ROAD MAP- 2 Strategic goal (Animal Feeding): To review certain measures of the current total feen ban when certain conditions are met, taking into account the reliable methods in place: Possible Gradual lifting of the Feed Ban Set up a tolerance level for PAP and MBM in feed for farmed animals Lifting feed ban provisions for non- ruminants (poultry, pig, fish): Allow interspecies use of MBM (e.g. Pig PAP & MBM to feed poultry) New validated test necessary!! MICROSCOPY OF ANIMAL COMPONENTS

7. WORKSHOP ON BSE CONTROL MEASURES- KIEV Porcine meat and bone meal MICROSCOPY OF ANIMAL COMPONENTS

8. WORKSHOP ON BSE CONTROL MEASURES- KIEV MICROSCOPY OF ANIMAL COMPONENTS SCIENTIFIC ASSESSMENT OF MBM/ PAP DETECTION METHODS

9. WORKSHOP ON BSE CONTROL MEASURES- KIEV MICROSCOPY OF ANIMAL COMPONENTS Community Reference Laboratory for Animal Proteins in Feedingstuffs. (CRL- AP) http://crl.cra.wallonie.be/ According to Aside the official light microscopy based method, other alternative techniques may be used for the detection of PAPs in feed : PCR (Polymerase Chain Reaction) Immunoassays NIRM (Near Infrared Microscopy) Combined NIRM - PCR approach These alternatives are however not yet official for the evident reason that most of these methods are still in development and hence not yet validated. The role of the EURL-AP is therefore also evaluating the potential of each analytical method.

10. WORKSHOP ON BSE CONTROL MEASURES- KIEV MICROSCOPY OF ANIMAL COMPONENTS Other Tasks: A) Coordination of Scientific Assessment: STRATFEED – EUROPEAN PROJECT G6RD 2000 CT 00414 Analytical methods for feed focusing an the detection of banned meat and bone meal B) Micrographs collection The largest repository of micrographs related to the detection of PAPs in feed, including more than 800 images

11. WORKSHOP ON BSE CONTROL MEASURES- KIEV MICROSCOPY OF ANIMAL COMPONENTS Micrographs collection This collection is also accessible to IAG- International Association for Feedingstuff Analysis members (public institution or private companies) on request:

12. WORKSHOP ON BSE CONTROL MEASURES- KIEV MICROSCOPY OF ANIMAL COMPONENTS STRATFEED – EUROPEAN PROJECT G6RD 2000 CT 00414 References for Analytical methods for feed focusing an the detection of banned meat and bone meal: Stratfeed project overview - Pierre Dardenne, CRA-W, BE Microscopic detection of animal proteins - Leo van Raamsdonk, RIKILT, NL PCR as tool to identify taxon-specific processed animal proteins - Gianfranco Brambilla, ISS, I Application of Near Infrared Spectroscopy (NIRS) to detection of Mammalian Meat and Bone Meal (MBM) in Feeds - Ian Murray, SAC, UK Review of the possibilities offered by the near infrared microscope (NIRM) and near infrared camera (NIR Camera) for the detection of MBM - Vincent Baeten, CRA-W, BE Counter Immuno Electrophoresis: Methodology for confirmation of positive results on mmunoassay and DNA based tests through detection of milk based proteins - Scott Reaney, Veterinary Laboratories Agency, UK Meat and bone meals in feed: Results from recent interlaboratory studies for the validation of methods and for the evaluation of the proficiency of laboratories - Christoph Von Holst, JRC-IRMM, BE

13. WORKSHOP ON BSE CONTROL MEASURES- KIEV MICROSCOPY OF ANIMAL COMPONENTS Fumière, O, Veys, P. Boix, A, Von Holst, C, Baeten, V et Berben, G. Methods of detection, species identification and quantification of processed animal proteins in feedingstuffs- 2009 Presently, optical microscopy remains the only reference method for the detection of PAPs to be applied for official control as required by Commission Directive 2003/126/EC. Other methods may be applied in addition to classical microscopy: – the detection of constituents of animal origin, – the detection of proteins from terrestrial animals in presence of fishmeal. PCR (Polymerase Chain Reaction), near infrared microscopy and imaging, as well as immunology Up to now, none of the techniques considered, enable a full implementation of the European Legislation (detection at low level, identification at species level and quantification of PAPs)

14. WORKSHOP ON BSE CONTROL MEASURES- KIEV Fumière, O, Veys, P. Boix, A, Von Holst, C, Baeten, V et Berben, G. Methods of detection, species identification and quantification of processed animal proteins in feedingstuffs- 2009 MICROSCOPY OF ANIMAL COMPONENTS

15. WORKSHOP ON BSE CONTROL MEASURES- KIEV Domenis, L., Squadrone, S., Marchis, D. et Abete, M (2009) OPTICAL MICROSCOPY Density of osteocyte lacunae Difficult to differentiate between mammalian and poultry bones Need to exam several characteristics such as colour and borders of the bone fragments, shape and density MICROSCOPY OF ANIMAL COMPONENTS

16. WORKSHOP ON BSE CONTROL MEASURES- KIEV Sampling preparation Domenis, L., Squadrone, S., Marchis, D. et Abete, M (2009) MICROSCOPY OF ANIMAL COMPONENTS

17. WORKSHOP ON BSE CONTROL MEASURES- KIEV Osteocyte lacunae features in chicken bones Domenis, L., Squadrone, S., Marchis, D. et Abete, M (2009) MICROSCOPY OF ANIMAL COMPONENTS

18. WORKSHOP ON BSE CONTROL MEASURES- KIEV Guidelines for scopes EA 2/05:2001 The scope of accreditation and consideration of methods and criteria for the assessment of scope in testing (to be reviewed and revised) EA 2/15 EA:2008 Requirements for the accreditation of flexible scopes ILAC G18:2010 Guideline for the formulation of scopes of accreditation of laboratories ACCREDITATION

19. WORKSHOP ON BSE CONTROL MEASURES- KIEV FLEXIBLE SCOPE Allows the laboratory not only to carry out the methods in use, but also to develop and validate tests in accordance with pre- established system and therefore to add methods within the defined limits to the scope of accreditation on the basis that their competence has been evaluated Assessment for flexible scopes is more demanding, both initially and ongoing Suitable for activity were development is intensive and changes are frequent ACCREDITATION

20. 1.- Legislation in place: Royal Decree 2257/ 2004, of 2 July, on official analysis methods for feed and feed materials Order PRE/1629/2004, of 2 June, including Annex with technical specifications 2.- National Reference Laboratory for Animal Feed Current Situation in Spain WORKSHOP ON BSE CONTROL MEASURES- KIEV MICROSCOPY OF ANIMAL COMPONENTS

21. WORKSHOP ON BSE CONTROL MEASURES- KIEV Control on marketing and trade, including import/export Control on premises, feed bussines, feed operators Farms control Animal Feed Control Plan R (EC) 882/2004 R (EC) 183/2006 National Reference Laboratory for Animal Feed MICROSCOPY OF ANIMAL COMPONENTS

22. WORKSHOP ON BSE CONTROL MEASURES- KIEV MICROSCOPY OF ANIMAL COMPONENTS Spain National Reference Laboratory for Animal Feed: Laboratorio Arbitral Agrario Ctra de La Coruña- MADRID http://www.mapa.es/es/alimentacion/pags/laboratorio/introduccion.htm Reference Laboratory for: Heavy Metals Residues of pesticides PAP / MBM controls Feed materials Plant protection products

23. THANK YOU FOR YOUR ATTENTION WORKSHOP ON BSE CONTROL MEASURES- KIEV