R.J. Margry, S.G.C. van den Hoven, J.C.H. Vaessen and J.M.H. van Doremalen.
CCL Nutricontrol, P.O. Box 107, 5460 AC Veghel, the Netherlands,
As a preventive measure to avoid the spread of Bovine Spongiform Encephalopathy (BSE), a feed ban had been introduced by the European Union (EC Regulation 999/2001). The Animal By-Product Regulation (EC) No.1774/2002 allows the use of products from category 3 materials for inclusion in feed, but this is subjected to strict rules, including an intra-species recycling ban. It is prohibited to feed animals with processed animal proteins (PAP’s) from the same species. Detection and identification of animal tissues in feed has therefore gained great interest.
At present, microscopy is still the only official method for detection of PAP’s in feed in the European Union.
Within the SAFEED-PAP project several methods are investigated for suitability of species identification. Besides (NIR) microscopy there is a need for techniques which can discriminate between species and which are less time consuming. The availability of analytical (screening) techniques for species identification would facilitate the lift of the feed ban. The suitability of several ruminant, porcine and poultry identification assays is tested with different reference materials. Some of this reference material (porcine soft material, porcine bone material, chicken soft material and chicken bone material) is processed by CCL in a dedicated steriliser under strict conditions (20 minutes at 133°C or 159°C; both pre- and post-pressure cooked).
Other reference material (bovine, ovine, porcine and avian carcase and muscle material) is processed by PDM Ltd (Doncaster, UK) at 133°C, 137°C, 141°C and 145°C. Furthermore, some ‘pure’ commercial available products are used as reference material. With these reference materials several mixtures are prepared, both in PAP and feed.
Two immunochemical methods (Reveal® kit of Neogen and MELISA-TEK™ of ELISA Technologies Inc.) and two PCR assays (methods of CRA-W, Gembloux, Belgium and of TNO, Zeist, the Netherlands), are promising and are extensive validated with (mixtures) of the reference materials. CCL uses the immunochemical methods for screening and the PCR method for confirmation.
The results show that the PCR assays and Reveal® and MELISA-TEK™ seem quite suitable for species identification, even after heating for twenty minutes at the highest temperatures. Especially, details of the MELISA-TEK™ screening method will be presented. A dilemma, due to the allowed (cattle) materials (i.e. milk products) in feed, will be shown.
As a solution, it is suggested to use analytical methods for control of PAPs, combined with administrative monitoring of compound feed. In general, the results show that these assays are quite promising. This will help acceptance of porcine and poultry PAP’s in aquafeed.
Keywords
Species identification, intra-species recycling ban, PCR, immunochemical assay, reference materials, Reveal, MELISA-TEK.