The recent TSE Roadmap II states that potential partial relaxation of the total feed ban will base on a stepwise approach supported by scientific bases. In this respect improvement of existing methods as well as the development of new ones are crucial for supporting any future amendment while still monitoring the potential re-emergence of BSE.

EURL-AP research activity in this area, in close collaboration with the NRLs, primarily focussed on:

• The improvement of the current light microscopic protocol
• Innovative combinations of methods with a view to the implementation of a future species-to-species feed ban

Revision of the official method

After an almost two years long working period, including a validation study implying the NRLs, the EURL-AP delivered by Mid April 2011 a revised protocol of Annex VI of EC regulation 152/2009 to DG Sanco. This revision was validated by an interlaboratory study performed in 2010 which demonstrated overall better performances. On 16th January 2013, Commission Regulation EU/51/2013 amending the former official method by the EURL-AP revision work was issued.

Major amendments included in this revision were:

• Standardization of sedimentation equipment,
• New sieving or grinding options,
• Optional use of stereomicroscope,
• Possibility to use the flotate instead of the raw material,
• Imposition of a minimum of 6 slides before declaring a sample as negative,
• Introduction of sequence diagrams for the observations,
• Mandatory repetition of analysis when few animal particles are detected,
• Result expression mode adapted.

The revised protocol does not longer include a quantification method. In point of fact the EURL-AP conducted with the participation of the NRLs several studies intended to improve the existing method of quantification. Although major improvements in accuracy and repeatability were obtained, the variability of results among different laboratories (reproducibility) is still not satisfying and hence no validation of any method could be achieved. The problem of PAPS quantification in feed remains inapplicable by light microscopy.

Combination of methods

“No method tells the whole truth.”

Regardless of whether analyses are carried out by light microscopy, by PCR, by NIRM or by screening methods such as immunoassays, none of the used method shall deliver the entire information necessary for a species-to-species feed ban. Therefore combination of methods – principally light microscopy and PCR as these are the most successful– is required. Such combination is mandatory for aquafeed analysis since 16th of January 2013.

Some few illustrations…

• Recently non-ruminant PAPs were authorised again in aquafeed. According to TSE Roadmap II, porcine PAPs could also be introduced in the future to poultry feed. Light microscopic analysis of such poultry feed containing porcine PAPs will reveal only the presence of terrestrial PAPs but will be unable to inform on the species of origin of the PAP used. Complementary information by PCR would indicate the porcine nature of the PAP. Furthermore PCR shall also reveal the presence or absence of prohibited poultry material which is prohibited to prevent intraspecific recycling.

Whether or not PCR is performed first would probably depend on the type of feed. It is certainly not justified for every feed type as in the next example.

• Dairy by-products are authorized in bovine feed. PCR performed on such feed shall react positively to a bovine or ruminant target, but shall not succeed in delivering information on the nature of the ingredient responsible for the signal. A second line light microscopic analysis on this feed will help to certify the absence of prohibited material as PAPS –as no bones, no cartilage and no muscles will be found. The feed can therefore be declared as fit for its purpose.

In the second example, light microscopic analysis would have been performed first without even the need for a complementary PCR test. Such option would spare time and finances.

• Sometimes official reports mention the presence of terrestrial material detected by light microscopy in fish meals. PCR tests shall in the future be able to confirm that this material originates from unintentional sea mammals by-catch instead of real terrestrial animals. In addition, as recently re-authorized, fish feed may aside fish meal as well contain terrestrial PAPs on the exclusion of ruminant ones. In case of absence of information related to the composition, a first test by light microscopy shall disclose the presence of terrestrial PAPs whereas a successive PCR testing will ascertain that those PAPs do not originate from ruminants hence enabling to declare the fish feed as fit for its utilization.