Scientific Opinion of the Panel on Biological Hazards. Adopted on 6 December 2007

Transcription

1 The EFSA Journal (2007), 596, 1-45 Transmissible Spongiform Encephalopathy (TSE) risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association 1 Scientific Opinion of the Panel on Biological Hazards (Question No EFSA-Q ) Adopted on 6 December 2007 PANEL MEMBERS Olivier Andreoletti, Herbert Budka, Sava Buncic, Pierre Colin, John D Collins, Aline De Koeijer, John Griffin, Arie Havelaar, James Hope, Günter Klein, Hilde Kruse, Simone Magnino, Antonio Martínez López, James McLauchlin, Christophe Nguyen-The, Karsten Noeckler, Birgit Noerrung, Miguel Prieto Maradona, Terence Roberts, Ivar Vågsholm, Emmanuel Vanopdenbosch. SUMMARY In its Opinion of 21 October 2004 on BSE risk from dissemination of brain particles in blood and carcass following stunning, the EFSA concluded that the brain damage caused by both penetrating and non-penetrating captive bolt stunning in cattle, as well as that caused by penetrating captive bolt in sheep can result in occurrence of central nervous system tissue in venous blood draining the head. The risk could however not be quantified. In its Opinion of 28 April 2005 on the assessment of the health risks of feeding of ruminants with fishmeal in relation to the risk of TSE the EFSA concluded that if there is any risk of TSE in fishmeal, this could arise from the mammalian feed being fed to fish which are then included in fishmeal or through fishmeal contaminated by Meat and Bone Meal (MBM). The risk of TSE in fish, either being fed directly or by amplification of infectivity is remote. Against this background, the European Commission has requested to the Scientific Panel on Biological Hazards to deliver a scientific opinion on a TSE risk assessment of the use of bovine blood in feeds for fish, in consideration of a report produced by the European Animal Protein Association (EAPA). The EFSA opinion considers that the EAPA report is well written and comprehensive. However, its qualitative approach does not fully take into account the uncertainties surrounding several of its risk parameters. Consequently, its conclusions may be overly optimistic. 1 For citation purposes: Opinion of the Scientific Panel on Biological Hazards on the request from the European Commission on a Transmissible Spongiform Encephalopathy risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association. The EFSA Journal (2007), 596, European Food Safety Authority, 2007

2 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association A human or animal health risk may arise if recycling of BSE-contaminated bovine SDRC occurs directly (bovine SDRC fed to cattle) or indirectly (fishmeal made from fish recently fed with BSE contaminated bovine SDRC given to cattle) because this would be equivalent to feeding cattle by-products to cattle (intra-species recycling). The assessment of the BSE related-risk of bovine SDRC from slaughtered bovine animals considered fit for human consumption to be included in aqua feed is theoretically feasible both semi-quantitatively and quantitatively by developing a probabilistic risk assessment model. However, key parameter limits of this model (i.e. endogenous bovine blood BSE infectivity and degree of contamination with CNS by current stunning and slaughter methods) can only be developed from expert opinion and judgement, as there is currently not experimental data available. Both the degree of uncertainty of this type of data (which would reduce the robustness of any risk estimates) and the extensive work that would be needed to produce such model makes its development unrealistic in the frame of this opinion. On the other hand and considering the current implementation of the EU feed-ban, the inclusion of bovine blood products in the authorized list of ingredients in fish feed would potentially limit the suitability of current available tools, to detect the presence of prohibited bovine by-products (i.e. SRM) Following this, the BIOHAZ panel recommends to develop and assess the outcome of a semiquantitative or quantitative risk model of the BSE risk of bovine SDRC employed in aqua feed. In order to enhance the robustness of that risk assessment with experimental data which is currently not available, it is further recommended to quantitatively evaluate different risk parameters. These would include the evaluation of the endogenous blood infectivity levels in incubating and terminally BSE affected cattle, the evaluation of the current allowed methods for cattle stunning for the potential to produce embolism and the quantitative assessment of the CNS contamination risk posed by different blood collection methods. Finally, a combination of tests capable of detecting, with a high level of sensitivity, the species and tissue origin of the animal proteins included in fish feed should be developed and validated. Key words: TSE, bovine blood, bovine spray dried blood cells, fish feed. The EFSA Journal (2007) 596, 2-45

3 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association TABLE OF CONTENTS Panel Members...1 Summary...1 Table of Contents...3 Background as provided by the European Commission Scientific knowledge Feed ban provisions...4 Terms of reference as provided by the European Commission...5 Acknowledgements...5 Assessment Introduction Scientific data and former assessments Former assessment on bovine blood safety BSE in fish Review of the EAPA report Risk Assessment on the TSE risk of Bovine SDRC in Feed for fish Ruminant feed cross contamination and environmental issues TSE endogenous infectivity in bovine blood Cross-contamination with possible TSE infected material Cross contamination with Central Nervous System tissue during slaughter Tests to identify the presence of CNS tissue in blood Detection tests for bovine blood cells and their limitations in fish feed Feasibility for a semi-quantitative risk assessment of SDRC from slaughtered bovine animals considered fit for human consumption to be included in aqua feed Essential elements for the development of a quantitative risk assessment of bovine spray dried red cells in feed for fish The BSE infectious load batch of bovine SDRC Number of infected animals entering the SDRC process (N) Infectious load per animal (I) Infectivity reduction by processing Use in Aqua Feed Conclusion on the feasibility for both a semi-quantitative and quantitative risk assessment 15 Conclusions and Recommendations...15 Documentation provided to EFSA...16 References...17 Appendices...21 Appendix I: Results of the control programmes of the feed ban in the EU member states...21 Appendix II: Farmed Fish Production Systems and the Fate of Sewage Water...22 Appendix III: Summary of research findings on TSE infectivity in blood of animals with TSE...26 Appendix IV: The different stages of the cattle slaughter process...31 Appendix V: Stunning methods and their relative effects on brain tissue embolism...33 Appendix VI: Tests to identify the presence of Central Nervous System tissue in blood...39 Appendix VII: Tests to identify the presence of bovine blood cells and their limitations in fish feed References from Appendix VII...42 Glossary / Abbreviations...43 The EFSA Journal (2007) 596, 3-45

4 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION Background Scientific knowledge In its Opinion of 21 October 2004 on BSE risk from dissemination of brain particles in blood and carcass following stunning, the EFSA concluded that the brain damage caused by both penetrating and non-penetrating captive bolt stunning in cattle, as well as that caused by penetrating captive bolt in sheep can result in occurrence of Central Nervous System (CNS) tissue in venous blood draining the head. The risk could however not be quantified. In its Opinion of 28 April 2005 on the assessment of the health risks of feeding of ruminants with fishmeal in relation to the risk of TSE the EFSA concluded that if there is any risk of TSE in fishmeal, this could arise from the mammalian feed being fed to fish which are then included in fishmeal or through fishmeal contaminated by Meat and Bone Meal (MBM). The risk of TSE in fish, either being fed directly or by amplification of infectivity is remote Feed ban provisions A ban on the feeding of mammalian meat and bone meal (MBM) to cattle, sheep and goats was introduced as of July This partial ban was extended to a total EU wide suspension on the use of processed animal protein in feeds for any animals farmed for the production of food on 1 January 2001 with some exceptions like the use of fish meal for non-ruminants. The use of blood products derived from non-ruminants is allowed for the use in feed destined for non-ruminants and fish. Report on a risk assessment as regards the use of bovine red cells (haemoglobin) in feeds for fish The European Animal Protein Association (EAPA) submitted a report prepared by EAPA and TSE expert, Dr. Ray Bradley, on a risk assessment as regards the use of bovine red cells (haemoglobin) in feeds for fish. In the report it is concluded that the TSE risk in spray-dried red cells derived from healthy cattle passed for human consumption is negligible. Evaluation of the overall situation by the Commission. The Communication from the Commission of 15 July 2005 on the future of TSE measures, the TSE roadmap, sets the strategic goal, regarding to the feed ban provisions. The starting point when revising the current feed ban provisions should be risk-based but at the same time taking into account the control tools in place to evaluate and ensure the proper implementation of this feed ban. The report elaborated by EAPA contains elements that could be relevant in relation to the revision of the feed ban provisions and in particular the semi-quantitative assessment of the risk of spray-dried red cells of bovine animals. Therefore it is appropriate to request an opinion of the European Food Safety Authority on the subject. The EFSA Journal (2007) 596, 4-45

5 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION The European Food Safety Authority (EFSA) is invited to provide an opinion on: The validity of the conclusions of the enclosed report. The topics that should be addressed in future studies on the subject if needed. The assessment of the BSE related risk, if feasible semi-quantitatively, of spray-dried red cells from slaughtered bovine animals considered fit for human consumption to be included in aqua feed. ACKNOWLEDGEMENTS The European Food Safety Authority wishes to thank the members of the Working Group for the preparation of this opinion: Olivier Andreoletti, Haluk Anil, Gilbert Berben, Herbert Budka, James Hope, Trond Storebakken and Emmanuel Vanopdenbosch (Chairman and Rapporteur). The BIOHAZ panel members would like to acknowledge Dr. Javier Polo for his availability when clarifying certain aspects related to the content of the EAPA report. ASSESSMENT 1. Introduction Since Bovine Spongiform Encephalopathy (BSE) was reported for the first time in 1986 in the UK, the European Union (EU) has developed a comprehensive set of risk reducing measures on transmissible spongiform encephalopathies (TSEs) in order to protect human health from BSE and to control and eventually eradicate TSEs in animals. That legislation has continuously been reviewed in the light of new scientific evidence, the evolution of the TSE situation and the practical implementation in the field. A ban on the feeding of mammalian meat and bone meal (MBM) to cattle, sheep and goats was introduced as of July This partial ban was extended to a total EU wide suspension on the use of processed protein in feeds for any animals farmed for the production of food. This key piece of legislation to protect human and animal health from the risk of BSE and other TSEs was adopted on 22 May This Regulation (EC) No 999/2001 of the European Parliament and of the Council (EC, 2001) lays down rules for the prevention, control and eradication of certain TSEs. One of the most effective risk reducing measures consisted of a total EU wide ban on the use of processed animal protein (PAP) in feeds for any animal farmed for the production of food, with some exceptions like the use of fishmeal in non-ruminants. This measure was introduced in January The use of blood products derived from non-ruminants is allowed for the use in feed destined for non-ruminants and fish. Since the implementation of the TSE Regulation in 2001, more than 60 million of adult bovine animals have been tested across the EU and around 7000 cases have been detected. A constant decline (about 30% per year) in the number of cases has been recorded: from 2167 cases in 2001 to around 320 in 2006 (EC, 2007). Out of this, only 27 cases were related to animals born after the start of the total feed ban as mentioned above. The EFSA Journal (2007) 596, 5-45

6 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association Feed microscopy is currently the only method officially endorsed by the European Commission to test for the presence of animal protein in feeds (EC, 2003). This method cannot discriminate within the mammalian species, up to the species level. However, recently new methods, i.e. (RT)-PCR, immuno-assays and Near Infrared (NIR) spectroscopy, are under development and validation, and could in principle identify the species composition of MBM. This assessment takes account of the general control measures in place and assumes the effectiveness of these controls in avoiding any possible cross-contamination, both deliberate and accidental. The implementation of the feed ban and national feed ban controls are audited by the Food and Veterinary Office of the European Commission documented in their reports. The results of the control programmes in the EU Member States are summarized in Appendix Scientific data and former assessments 2.1. Former assessment on bovine blood safety The SSC opinion on The safety of ruminant blood with respect to TSE risks adopted on April 2000 (SSC, 2000) stated that no infectivity was demonstrated in blood from BSE affected cattle (both naturally and experimentally infected). Regarding ruminants in general (including small ruminants), it was assumed that low levels of TSE infectivity could be present in blood. However, it was considered to be more relevant the risk of contamination with CNS tissue by employing some stunning methods and during processing (e.g. carcass splitting). Nevertheless, pooled blood collected from healthy slaughter cattle passed fit for human consumption was considered to potentially carry low levels of infectivity. It was also advised that intraspecies recycling of ruminant blood and blood products should be avoided. In this SSC opinion of 2000 on the safety of ruminant blood (SSC, 2000) the Working Group considered that the most important aspect of the risk assessment related to brain tissue contamination. It was proposed that the risk assessment needed to be made on a regional base. Three were the levels at which the assessment was done: (1) Slaughterhouse, (2) Geographical BSE risk and surveillance and (3) Use of blood. At those levels, different factors that would influence the risk are here included in table 2, as extracted from that opinion. The EFSA Journal (2007) 596, 6-45

7 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association Table 2. Qualitative risk assessment of TSE infectivity in bovine blood (SSC, 2000). Increased risk Decreased risk high incidence of BSE in slaughter animals animals > certain age slaughtered invasive stunning devices small slaughterhouses blood frequently collected low incidence of BSE in slaughter animals animals < certain age slaughtered non-invasive stunning devices large slaughterhouses blood infrequently collected amount of blood pooled * amount of blood pooled * large amount used for consumption mild processing of blood products feeding to same species (no species barrier) small amounts used for consumption severe processing of blood products feeding to humans/different species (species barrier) * A high degrees of dilution would result in a lower residual infectivity per unit of volume, but may also imply that a much higher number of individuals be exposed to (lower) infectivity. In terms of possible infections, the outcome may eventually appear to be the same (see also the Pre-opinion of 2-3 March 2000 of the Scientific Steering Committee on Oral exposure of humans to the BSE agent: infective dose and species barrier). In its Opinion of 21 October 2004 on BSE risk from dissemination of brain particles in blood and carcass following stunning (EFSA, 2004), the EFSA concluded that brain damage caused by both penetrating and non-penetrating captive bolt stunning in cattle, as well as caused by penetrating captive bolt in sheep can result in occurrence of CNS tissue in venous blood draining the head. The risk could however not be quantified BSE in fish The possibility of clinical or sub-clinical infection of fish and the risk of residual infectivity after exposure to BSE contaminated feed has been assessed in an opinion of the Scientific Steering Committee (SSC) as well as in an EFSA opinion. In its Opinion of 6-7 March 2003 on the feeding of wild fishmeal to farmed fish and recycling of fish with regard to the risk of TSE (SSC, 2003), the SSC addressed the question whether the historical practice of feeding Mammalian MBM and other mammalian products and the common practice of intra-species and intra-order recycling via feed could enable mammalian TSE agents to establish themselves in fish and for species adaptation of such agents to occur. The SSC concluded that on the basis of limited available research results there was no evidence of any such risk existing. However, some theoretical risks were identified, linked to feeding possibly TSE-contaminated feeds to animals currently believed to be not susceptible, including fish. The SSC therefore considered in general that fish should not be fed with potentially TSE infected feed and that sourcing of fish by-products (including for their use in fish-derived feed) should not be performed from fish that have been exposed to potentially infected feed. With regard to the appropriate treatment of fish materials, further background information is provided in the Opinion on Fallen stock by the Scientific Steering Committee (SSC, 1999) and the Scientific Committee on Animal health and Welfare (SCAHAW, 2003). The EFSA Journal (2007) 596, 7-45

8 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association In the EFSA Opinion of 24 th January 2007 on the assessment of the health risks of feeding of ruminants with fishmeal in relation to the risk of TSE (EFSA, 2007a) it was concluded that if there is any risk of TSE in fishmeal, this could arise from the mammalian feed being fed to this fish or through fishmeal contaminated by Meat and Bone Meal (MBM). The risk of TSE in fish, either being fed directly or by amplification of infectivity was considered to be remote. In addition, it was stated that although a huge progress is made in tests used for the detection of MBM in feed and that progress in tests developed and the combination of different tests now allow a better detection and differentiation of MBM up to the species level, there is still no 100 % guaranteed method available. 3. Review of the EAPA report In general, the EAPA report summary suggests that since BSE cases are reduced and effective control measures have been in place for a considerable period, risks to human health from animal products have been reduced to a minimum. It is further suggested that since the use of blood products from non-ruminant animals has been relaxed, in line with that, bovine animal products including SDRC can now be safely used. In particular, the EAPA report claims that its assessment, described as a risk analysis, shows negligible threat to human health from potential use of SDRC. As a result, it is proposed that bovine SDRC be used as a protein supplement in fish feed. It is also pointed out that introduction of the use of bovine SDRC can have economic benefits to the industry as well as protect the environment. It is claimed that this can be achieved by reducing disposal costs of bovine blood, reduce CO 2 emissions and also by providing cheap protein feed for fish farming. Even though it is stated in page 26 of the EAPA report, it should also be highlighted here that the terminology spray dried red cells does not reflect entirely the nature of this product, as also other blood cells and platelets are present. Thus, in page 26 of the report it is described that SDRC is actually largely a mixture of red and white cells. To keep nomenclature in line with that of the EAPA report, the use of SDRC will be maintained throughout this document, but with the above mentioned reservations regarding its inherent meaning. The EAPA report does not refer to the available EFSA report on stunning and TSE risks (EFSA, 2004). While the EAPA report qualifies the risk of CNS embolism from non- penetrating captive bolt stunning as negligible, the EFSA 2004 Opinion did not exclude a risk of CNS embolism from both penetrating and non-penetrating captive bolt stunning. Only the prevalence of embolism after the use of penetrating or non-penetrating guns is known, but not of neither the use of pithing nor air guns. However, this lack of knowledge is of little relevance as neither pithing nor air gun stunning is currently allowed in the EU. The EAPA report further claims that only blood is collected. This may be achievable with the socalled Hollow Knife system (page 44). However, given the variety of blood collection methods used in slaughterhouses (see Annex 2 to this opinion), the collected blood could be contaminated with brain and other CNS tissues. Such material would present an increased risk 2. Sealing the captive bolt stunning hole before bleeding out would also decrease the risk of possible cross contamination. 2 Blood collected in a tank or trough by leaving the animal after stunning and sticking to loosely bleed out could get contaminated with some minor amount of CNS tissue (i.e. cerebral tissue), which may leak from the hole made in the frontal region of the head by the penetrative captive bolt stunning gun. Similarly and in very small traditional abattoirs, carcase splitting could be carried out close to the bleeding area. Aerosols originated during this operation may contaminate with some minor amount of CNS tissue (i.e. spinal cord tissue) the collected blood. The EFSA Journal (2007) 596, 8-45

9 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association In sum, the EAPA report concludes that the risk from currently EU permitted stunning methods is negligible because of the measures that have been taken. However, the EFSA opinion of 2004 (EFSA, 2004) and more recent data show that some cross-contamination during stunning could not be excluded. The report states that blood collected from a living animal that passes ante and post mortem examination (and if > 30 months old a rapid test) could be considered to present a TSE negligible risk". However, the currently employed rapid tests do only detect infectivity from around 80% of the incubation period onwards (EFSA, 2007b). In regard to detection, the EAPA report states (page 11) that it is difficult to differentiate between fish meal and other processed animal proteins (PAP). This is not true, as the difficulty stems more from the fact that the presence of high amounts of fish meal makes it more difficult to detect low amounts of other PAP(s) (Gizzi et al., 2004) by using microscopy, which is the only officially recognised detection method (EC, 2003). The EAPA report clearly stresses (page 8) that the availability of effective methods to detect PAP in animal feed is very important, particularly with respect to prohibited ruminant proteins. However, the EAPA report does not consider the fact that reintroducing bovine spray dried blood will make interpretation of the detection results with regard to the origin of bovine proteins in fish feed more difficult. The EAPA report considers that the available tests for detection of PAP(s) work well when monitoring compliance with current legislation. However, this is only true in the context of the now existing bans and restrictions. If one of these prohibitions is lifted, the impact on detection should be assessed and this was completely disregarded in the EAPA report. It is for instance true, as stated at page 14 of the EAPA report, that Polymerase Chain Reaction (PCR) can detect the animal species but once bovine DNA is detected it is impossible to know if it is coming from spray dried blood cells that includes DNA containing leucocytes or from unauthorized bovine material (i.e. SRM). Several sets of data indicate that the conditions applied in the manufacturing process of bovine SDRC is unlikely to result in any significant reduction in the titre of TSE infectivity (page 45 of the EAPA report; SSC, 2000). The exposure of the product to 133 C 20 min 3 bar standard, which has been shown to reduce infectivity of at least 2 log 10, is not be technically possible for the production of bovine SDRC as it would affect the technical and functional properties of the product. 4. Risk Assessment on the TSE risk of Bovine SDRC in Feed for fish In considering the first bullet point of the mandate on the validity of the conclusions of the EAPA report the BIOHAZ Panel focused on the BSE risk of using SDRC as aqua feed. The Panel did not consider the scientific evidence in the EAPA report that is used to argue for its benefits in reducing disposal costs of bovine blood, in reducing CO 2 emissions and in providing cheap protein feed for fish farming Ruminant feed cross contamination and environmental issues Avoiding and limiting the risk of intraspecies recycling remain a central piece in the current EU policy for preventing BSE and other TSE(s) propagation in farm animals. As stated in the EFSA 2007 Opinion on Certain Aspects related to Feeding of Animal Proteins to Farm Animals (EFSA, 2007c), the transmission of BSE through small PAP quantities cannot be excluded. In The EFSA Journal (2007) 596, 9-45

10 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association the case of blood even if infectivity can certainly be considered extremely low, and would represent a negligible risk for human (species barrier), its potential ability to transmit BSE to cattle (no species barrier) at an extremely low level cannot, in the current state of knowledge, be ruled out. This may result from 1. The possibility of cattle been fed with proteins from bovine origin (i.e. intraspecies recycling). 2. The potential future use of fishmeal for feeding ruminants which could include byproducts from fish harvested after been fed (directly or indirectly) bovine spray dried red cells. Indirect feeding may occur when feed pellets employed in open systems 3 are eaten by opportunistic wild fish. Fishmeal is not currently employed in feeds for ruminants however Reg. 1923/2006 foresees the possibility for the EC to allow the use of fishmeal for young ruminants. 3. The environmental fate of sewage resulting from fish production (particularly in open systems) (Sara et al., 2004), as a percentage of the fish feed and fish excreta may reach water torrents and the fluvial environment. Further details on the farmed fish production systems and fate of sewage are provided in Appendix II TSE endogenous infectivity in bovine blood Data concerning the presence of TSE infectivity in blood or blood components from naturally or experimentally infected animals are summarized in appendix III. PrP c is expressed in various blood components from mammals including plasma, platelets and nucleated cells (MacGregor et al., 1999; Barclay et al., 2002; Thackray et al., 2005, 2006). In experimentally infected rodents PrP Sc has been detected after PMCA amplification of blood from incubating and terminally affected individuals (Castilla et al., 2005; Saá et al., 2006; Chang et al., 2007; Murayama et al., 2007). However, recently made available data suggested that PMCA could allow PrP Sc detection in tissues showing no infectivity after homologous bioassay (Green et al., 2007; Deleault et al., 2007). To date, low infectivity titre has been reported in blood from naturally affected TSE individuals (i.e. natural sheep scrapie), including nvcjd in human, and in numerous experimental TSE models. All these models concur in considering that infectivity is mainly associated to nucleated blood circulating cells and in lower proportion to plasma. In cattle BSE no infectivity associated to blood was reported after: a. inoculation in RIII mice of blood and blood components (Bradley, 1999). (Wells et al., 1998, 2005). b. inoculation of blood in cow (Wells et al., 1998, 2005). c. inoculation of blood to Tg Bov mice (Espinosa et al., 2007; Buschmann and Groschup, 2005). However data that are available are subjected to limitations, because: 3 In open farm fish systems fish are reared into cages, tanks or raceways directly into the water source (e.g. lakes, open sea). The EFSA Journal (2007) 596, 10-45

11 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association (i) existence of a species barrier in RIII mice that could limit detection of low infectious titre; (ii) low number of samples that were tested in homologous PrP model (cow and Tg Bov); (iii) volume consideration related to blood (low infectivity in large volume) (Wells et al.,1998, 2005). In conclusion, presence of low endogenous infectious titre in BSE affected blood cannot currently be ruled out. In any case, this infectious titre would be negligible when compared to CNS contamination Cross-contamination with possible TSE infected material Cross contamination with Central Nervous System tissue during slaughter The process of cattle slaughtering comprises a series of steps as described in Appendix IV. The SSC opinion of 2002 on the risk of dissemination of brain particles into the blood and carcass when applying certain stunning methods (SSC, 2002), considered the risk of CNS embolism associated with penetrating captive bolt stunning with air injection, as well as the risks associated with penetrating stunning followed by pithing, as particularly high. Following initial recommendations from the Scientific Steering Committee (SSC, 1997) and from the Scientific Committee of Veterinary Measures relating to Public Health (SCVMPH, 1998) and concurrent research findings (Anil, 1999) the use of these stunning methods by the meat industry was already banned following EU legislation from 2000 (EC, 2000). Therefore, the EFSA 2004 opinion did not re-consider the above mentioned methods (EFSA, 2004). A number of studies showed that there was a risk of embolism associated with both penetrative and non-penetrative captive bolt stunning, as presented in the 2004 EFSA opinion on BSE risk from dissemination of brain particles in blood and carcass following stunning. Different stunning methods (Mechanical, Electrical and Gas stunning methods) have been considered and reviewed in this opinion. Relevant details are given in Appendix V Tests to identify the presence of CNS tissue in blood The EFSA opinion on BSE risk from dissemination of brain particles in blood and carcass following stunning (EFSA, 2004) already reviewed different detection methods for the assessment of dissemination of CNS constituents as markers of presence of brain tissue in blood in particular. However, scientific literature does not document how processing of blood cells in the spray-drying process might affect results of the methods, that is why presently these techniques can only be considered in the early steps of the process, the more as, for some of these markers, the stability of the immuno-reactivity was checked only for some days and is presumed to decline afterwards due to proteolytic activity or to oxidation (Schmidt et al., 1999). Moreover, due to the low content of blood products in fish feed (from about 5 % in juveniles to about 10 % in adults (Hertrampf and Piedad-Pascual, 2000; Johnson and Summerfelt, 2000)) these methods may not be sensitive enough. Different methods (Glial Fibrillary Acidic Protein (GFAP) ELISA, Syntaxin 1B ELISA and NSE (neural enolase 2) and S100 ELISA) were considered and relevant details are given in Appendix VI. The EFSA Journal (2007) 596, 11-45

12 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association Detection tests for bovine blood cells and their limitations in fish feed Presence of bovine spray-dried blood cells in fish feed could be detected by employing several methods. However, the inherent problem is determining exactly the species and tissue of origin of the protein source. Effectiveness and limitations of the several detection techniques to that purpose are reviewed in Appendix VII. By combining several of the reviewed methods it seems feasible to check whether bovine SDRC and/or blood products of bovine origin were used in fish feed manufacturing. Inclusion of bovine blood products in the authorized list of ingredients in fish feed would potentially limit the suitability of current available tools to detect for the presence of prohibited bovine by-products (i.e. SRM). The results of PCR and other DNA based methods would be particularly prone to misinterpretation if bovine SDRC, which includes DNA containing leucocytes, was included in feed Feasibility for a semi-quantitative risk assessment of SDRC from slaughtered bovine animals considered fit for human consumption to be included in aqua feed Previous EFSA opinions have dealt with the quantitative assessment of the risk posed by certain bovine by-products (EFSA, 2005a; EFSA, 2005b). For those particular assessments, several inherent elements related to the particular product assessed were identified and (where possible) quantified, in order to finally express the risk of exposure in units of BSE infectivity Essential elements for the development of a quantitative risk assessment of bovine spray dried red cells in feed for fish The essential elements or factors that need to be taken into account when approaching the development of a quantitative risk assessment (QRA) of SDRC from slaughtered bovine animals considered fit for human consumption would include: (1) The infectious load of cattle by-products (blood). (2) Assumptions regarding the total infectious load of cattle by-products (blood) annually entering a country s recycling chains. i. The BSE prevalence (detected numbers of BSE positives). ii. The ratio of sub-clinical non-detected to detected BSE positives. iii. Infectious load per animal. iv. Infectious load and age. v. Summary overview of the scenarios of total infectious load per country. (3) Assumptions regarding the yield of by-products (blood) per animal. (4) Possible (Contaminating) sources of BSE infectivity in cattle tissues and byproducts (Blood). i. Possible (contaminating) sources of BSE infectivity in cattle tissues and by-products (blood). ii. (5) Batch sizes. Risk scenarios for the possible inclusion of infection in raw materials. The EFSA Journal (2007) 596, 12-45

13 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association i. Number of animals per batch. ii. iii. Yield of SDRC per batch. Probability of material from an infected animal being present in a batch and number of infected cattle per batch. (6) Infectivity reduction by processing. (7) Fish consumption of bovine SDRC and environmental residues. The BSE infectious load per batch of bovine SDRC is the product of the number of infected animals entering the batch process (N) and the infectious load per animal (I). N is dependant on the geographical BSE risk of the country providing the cattle and the strength of its surveillance network while I is a function of (a) the volume of blood per animal, (b) its endogenous infectivity titre, and (3) the chance of CNS contamination. The exposure risk could then be estimated from this by considering how this infectious load might be influenced by processing and how its use (aqua feed) might disseminate infectivity into the food chain and the wider environment The BSE infectious load batch of bovine SDRC Number of infected animals entering the SDRC process (N) The BSE prevalence in the cattle population is the major risk determinant in assessing the quantitative effects of feeding bovine SDRC to fish. If BSE is absent from the cattle population then there is no risk in feeding bovine SDRC to fish. All EU MS have moderate risk status with respect to BSE and current surveillance continues to show a decreasing prevalence of disease. Since the implementation of the TSE Regulation in 2001, more than 60 million of adult bovine animals have been tested across the EU and around 7000 cases have been detected. A constant decline (about 30% per year) in the number of cases has been recorded: from 2167 cases in 2001 to around 320 in 2006 (EC, 2007). Out of this, only 27 cases were related to animals born after the start of the total feed ban as mentioned above. Ante mortem health inspections should exclude visibly sick animals entering the batch process and various scenarios can be modelled to estimate the probability of a pre-clinical BSE-infected animal entering the SDRC batch process. This would require the latest data from EU BSE surveillance and information on the number of animals used to produce each batch of SDRC. In the 2007 EFSA Opinion on the likelihood of the infectivity in SRM derived from cattle at different age groups estimated by back calculation modelling, the BSE infectivity is reasonably assumed to be present at approximately 80% of the incubation period. The number of cattle infected with BSE is likely to continue to decline, and only very rare cases are born after Cases detected by active surveillance may be closer to clinical onset than previously estimated (EFSA, 2007b), so the assumption on the number of infectious preclinical cases is accordingly to be decreased Infectious load per animal (I). This would depend on 3 factors: (a) Volume of blood per animal. The EFSA Journal (2007) 596, 13-45

14 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association The volume of blood in an adult cow is ~ 60 ml/kg, and the weight of the cow can vary up to 700 kg. An upper estimate of total yield would therefore be in the region of 40 litres (mean 20 litres; range 10-30). The accuracy of these estimates can be checked with data from production sites of SDRC. (b) Endogenous infectivity titre in blood from a BSE-infected bovine. No infectivity has been detected in blood from a cow clinically-affected by BSE but, as discussed above, the presence of levels of infectivity below the limit of detection (LOD) of current bioassays cannot be ruled out. For the purposes of a semi-quantitative risk assessment, some upper limit of this LOD, and its confidence intervals, could be made from published data (Buschmann and Groschup, 2005; Espinosa, 2007). In rodent and sheep models data available (Andreoletti et al., 2007; Brown et al., 1998, Cervenakova et al., 2003, Hunter et al., 2002) on the titre of infectivity within blood estimate a concentration between 1 and 10 intra-cerebral (ic) LD 50 units per ml. Assuming a similar scenario in cattle, a possible range of BSE infectivity would represent 1000 to ic LD 50 units per litre. This would be equivalent to between 0.1 and CoLD 50 per litre of cattle blood 4. (c) Chance of CNS contamination of blood. The probability of CNS contamination of blood depends on the stunning/killing practices and procedures at individual slaughterhouses and this may vary widely. No quantitative measure of this contamination by accident, or the result of embolism, could be made in previous risk assessments and no data have been collected to our knowledge to allow an estimate to be made. Similar deficits in knowledge were bridged previously by use of expert guesses (EFSA, 2005a); for example, we could assume a maximum contamination of blood with CNS of 10 mg per litre (mean, 1 mg/l; range, mg/L). Combining this value in a probabilistic risk assessment, with the likelihood of the co-incidence of contamination and an infected cow (see section 4.4.1), could give probability distribution curves of the degree of CNS contamination of blood with BSE infectivity Infectivity reduction by processing The process of spray-drying blood or an enriched red blood cell fraction of blood is not expected to significantly reduce any levels of endogenous or exogenous infectivity in this product (Taylor et al., 1998; Lipscomb et al., 2007) Use in Aqua Feed Several considerations have to be taken here: There is currently no evidence that fish or other non-mammalian species can replicate and amplify prions originating in cattle or other mammalian species. Therefore, no amplification factor needs to be introduced into a semi-quantitative risk assessment. However, it should be noticed that if recycling of BSE contaminated bovine SDRC occurs directly (bovine SDRC fed to cattle) or indirectly (fishmeal produced from fish 4 LD 50 units are used here rather than ID 50 units of infectivity because of the relative difficulty in determining an in vivo TSE infection, rather than onset of clinical disease (and death) following exposure to TSE agents. Most titres of TSE infectivity are usually quoted in LD 50 units because of this practical limitation. The EFSA Journal (2007) 596, 14-45

15 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association recently fed with BSE contaminated BSDRC fed fish) there is no species barrier (cattle intra-species recycling). Not all of the SDRC product used in aqua feed will actually be eaten: The environmental fate of sewage resulting from fish production (particularly in open systems) (Sara et al., 2004), as a percentage of the fish feed and fish excreta may reach water torrents and the fluvial environment. Further details are given in Appendix II. The level of this environmental contamination may be gauged from data on the tonnage of SDRC fed to captive marine animals and estimates of its utilisation (e.g., weight gain in fish). This may allow an estimate of the non-consumed residue and the potential amount of BSE infectivity it represents as contamination of the immediate environment of the fish pen, etc Conclusion on the feasibility for both a semi-quantitative and quantitative risk assessment The assessment of the BSE related-risk of bovine SDRC from slaughtered bovine animals considered fit for human consumption to be included in aqua feed is theoretically feasible both semi-quantitatively and quantitatively by developing a probabilistic risk assessment model, which would include multiple parameter limits. The resultant risk would ideally be expressed in CoID 50 units (Cattle oral infectious dose 50% probability) per unit weight of bovine SDRC product. Currently, key parameter limits such as the endogenous bovine blood BSE infectivity and the degree of contamination with CNS by current stunning and slaughter methods are unknown. This lack of data could be overcome by the use of expert opinion and judgement, i.e. not collected experimentally. However, it is acknowledged that the inherent degree of uncertainty of this type of data would reduce the robustness of any risk estimates. Extensive work would also be needed to produce such model and that makes its development unrealistic in the frame of this opinion. CONCLUSIONS AND RECOMMENDATIONS CONCLUSIONS The EAPA report is well written and comprehensive. However, its qualitative approach does not fully take into account the uncertainties surrounding several of its risk parameters. Consequently, its conclusions may be overly optimistic. A human or animal health risk may arise if recycling of BSE-contaminated bovine SDRC occurs directly (bovine SDRC fed to cattle) or indirectly (fishmeal made from fish recently fed with BSE contaminated bovine SDRC given to cattle) because this would be equivalent to feeding cattle by-products to cattle (intra-species recycling). The production technology employed for the manufacturing of SDRC as described in the EAPA report would be unlikely to reduce BSE infectivity if present. The assessment of the BSE related-risk of bovine SDRC from slaughtered bovine animals considered fit for human consumption to be included in aqua feed is theoretically feasible both semi-quantitatively and quantitatively by developing of a probabilistic risk assessment model. However, key parameter limits of this model (i.e. endogenous bovine blood BSE infectivity and degree of contamination with CNS by current stunning and slaughter methods) can only be developed from expert opinion and judgement, as there is The EFSA Journal (2007) 596, 15-45

16 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association currently not experimental data available. Both the degree of uncertainty of this type of data (which would reduce the robustness of any risk estimates) and the extensive work that would be needed to produce such model makes its development unrealistic in the frame of this opinion. Inclusion of bovine blood products in the authorized list of ingredients in fish feed would potentially limit the suitability of current available tools, to detect for the presence of prohibited bovine by-products (i.e. SRM). The results of PCR and other DNA based methods would be particularly prone to misinterpretation if bovine SDRC, which includes DNA containing leucocytes, was included in feed. RECOMMENDATIONS To develop and assess the outcome of a semi-quantitative or quantitative risk model of the BSE risk of bovine SDRC employed in aqua feed. To evaluate the endogenous blood infectivity levels in incubating and terminally BSE affected cattle, in order to provide a quantitative estimate of this source of blood infectivity as a parameter for the quantitative risk model. To evaluate the potential of currently allowed methods for cattle stunning to produce embolism, in order to provide a quantitative estimate of this source of blood infectivity as a parameter for the quantitative risk model. To assess in a quantitative way the risk of CNS contamination posed by different blood collection methods. To develop and validate a combination of tests capable of detecting with a high level of sensitivity, the species and tissue origin of the animal proteins included in fish feed. DOCUMENTATION PROVIDED TO EFSA Annex 1: The case for reintroducing spray-dried red cells (SDRC) from cattle passed fit for human consumption into the diet of farmed fish in the European Union. March Submitted by Ray Bradley and Javier Polo, from The European Animal Protein Association (EAPA). The EFSA Journal (2007) 596, 16-45

17 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association REFERENCES Andreoletti, O., Morel, N., Lacrous, X. Simon, S, Mathey, J., Lantier, I., Rouillon, V., Delmas, J.M., Weissbecker, J.L., Corbière, Fl, Simmons, H., Schelcher, F., Lantier, F. and Grassi, J Dynamics and Distribution of Infectivity in Sheep Blood. Prion 2007 Congress. Edinburgh, 26th, 27th and 28th September Anil, M.H., Love, S., Williams, S., Shand, A., McKinstry, J.L., Helps, C.R., Waterman-Pearson, A., Seghatchian, J. & Harbour, D.A Potential contamination of beef carcasses with brain tissue at slaughter. Vet. Rec., 145(16): Barclay, G. R., Houston, E. F., Halliday, S. I., Farquhar, C. F. and Turner, M. L Comparative analysis of normal prion protein expression on human, rodent, and ruminant blood cells by using a panel of prion antibodies. Transfusion 42 (5): Bradley, R BSE transmission studies with particular reference to blood. Dev Biol Stand (99): Brown, P., Rohwer, R.G., Dunstan, B.C., MacAuley, C., Gajdusek, D.C., Drohan, W.N The distribution of infectivity in blood components and plasma derivatives in experimental models of transmissible spongiform encephalopathy. Transfusion 38(9): Buschmann, A. and Groschup, M. H Highly Bovine Spongiform Encephalopathy: Sensitive Transgenic Mice Cornfirm the Essential Restriction of Infectivity to the Nervous System in Clinically Diseased Cattle. The Journal of Infectious Diseases, 192: Castilla, J., Saá, P. and Soto, C Detection of prions in blood. Nature Medicine, (11): Cervenakova, L., Yakovleva, O. and McKencie, C Similar levels of infectivity in the blood of mice infected with human-derived vcjd and GSS strains of transmissible spongiform encephalopathy. Transfusion 43(12): Chang, B., Cheng, X., Yin, S., Pan, T., Zhang, H., Wong, P., Kang, S-C, Xiao, F., Yan, H., Li, C., Wolfe, L. L., Miller, M., Wisnewski, T., Greene, M. W. and Syl, M.S Test for Detection of Disease-Associated Prion Aggregate in the Blood of Infected but Asymptomatic Animals. Clinical and Vaccine Immunology, 14, Deleault, N. R., Harris, B. T., Rees, J. R. and Supattapone, S Proceedings of the Nationall Academy of Science, USA. 104 (23), EC, Commission Decision 2000/418/EC regulating the use of material presenting risks as regards transmissible spongiform Encephalopathies. Official Journal of the European Communities, L 1: EC, Regulation (EC) No 999/2001 of the European Parliament and of the Council of 22 May 2001 laying down rules for the prevention, control and eradication of certain transmissible spongiform encephalopathies. Official Journal of the European Union 147: EC, Commission Directive 2003/126/EC of 23 December 2003 on the analytical method for the determination of constituents of animal origin for the official control of feedingstuffs. Journal Volume (Issue): The EFSA Journal (2007) 596, 17-45

18 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association EC, Report on the monitoring and testing of ruminants for the presence of Transmissible Spongiform Encephalopathy (TSE) in the EU in EFSA, Opinion on BSE risk from dissemination of brain particles in blood and carcass following stunning. The EFSA Journal (123): EFSA, 2005a. Opinion on the quantitative risk assessment of the animal BSE risk posed by meat and bone meal with respect to the residual BSE risk. The EFSA Journal, 257: EFSA, 2005b. Opinion on the assessment of the human and animal BSE risk posed by tallow with respect to residual BSE risk. The EFSA Journal, 221: EFSA, 2007a. Opinion on health risks of feeding of ruminants with fishmeal in relation to the risk of TSE. The EFSA Journal (443): EFSA, 2007b. Opinion of the Scientific Panel on Biological Hazards on the assessment of the likelihood of the infectivity in SRM derived from cattle at different age groups estimated by back calculation modelling. The EFSA Journal (476): EFSA, 2007c. Opinion on Certain Aspects related to Feeding of Animal Proteins to Farm Animals. The EFSA Journal (576): Espinosa, J. C., Morales, M., Castilla, J., Rogers, M. and Torres, J. M Progression of prion infectivity in asymptomatic cattle after oral bovine spongiform encephalopathy challenge. Journal of General Virology, 88: Gizzi, G., von Holst, C., Baeten, V., Berben, G. and van Raamsdonk, L Determination of processed animal proteins, including meat and bone meal, in animal feed. J AOAC Int 87 (6): Green, K. M., Castilla, J., Seward, T. S., Soto, C. and Telling, G. C Accelerated Studies of Interspecies Prion Transmission Using Combined Transgenic and PMCA Technologies. Presentation at the Prion 2007 congress. Edinburgh, 26, 27 and 28 September Hertrampf, J. W. and Piedad-Pascual, F Handbook of ingredients for aquaculture feeds. 624 pp. New York: Springer Publishing Company. ISBN Hunter, N., Foster, J., Chong, A., McCutcheon, S., Parnham, D., Eaton, S., MacKenzie, C., and Houston, F Transmission of prion diseases by blood transfusion. Journal of General Virology 83: Johnson, J.A. and Summerfelt, R.C Spray-dried blood cells as a partial replacement for fishmeal in diets for rainbow trout Orcorhynchus mykiss. Journal of the world aquaculture societ, 31 (1): Lipscomb I.P., Pinchin H., Collin R., Keevil C.W Effect of drying time, ambient temperature and pre-soaks on prion-infected tissue contamination levels on surgical stainless steel: concerns over prolonged transportation of instruments from theatre to central sterile service departments. J Hosp Infect 65(1):72-7. The EFSA Journal (2007) 596, 18-45

19 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association MacGregor I, Hope J, Barnard G., Kirby L, Drummond O Application of a time-resolved fluoroimmunoassay for the analysis of normal prion protein in human blood and its components. Vox Sang 77: Murayama, Y., Yoshioka, M,, Okada, H., Takata, M., Yokoyama, T. and Mohri. S Urinary excretion and blood level of prions in scrapie-infected hamsters. Journal of General Virology, 88, Saá, P., Castilla, J. and Soto, C Presymptomatic detection of prions in blood. Science, 5783 (313): Sara, G., Scilipoti, D., Mazzola, Z. and Modica, A Effects of fish farming waste to sedimentary and particulate organic matter in a southern Mediterranean area (Gulf of Castellammare, Sicily): a multiple stable isotope study (d13c and d15n). Aquaculture, 234, SCAHAW, Report of the Scientific Committee on Animal Health and Welfare on the use of fish by-products in aquaculture. Schmidt, G. R., Hossner, K. L., Yemm, R. S., Gould, D. H. and O'Callaghan, J. P An enzyme-linked immunosorbent assay for glial fibrillary acidic protein as an indicator of the presence of brain or spinal cord in meat. J Food Prot 62 (4): SCMVPH, Opinion of the Scientific Committee on Veterinary Measures relating to Public Health adoptend on the 17 th of February 1998 on the safety of slaughter practices and methods : risk of spread of BSE infectivity through cross contamination of different tissues by using pneumatic stunning during the slaughtering process of ruminants. SSC, Summary minutes of the meeting of the Scientific Steering Committee of the 8th and 9 th of December SSC, Opinion on the risks of non conventional Transmissible agents conventional infectious agents or other hazards such as toxic substances entering the human food or animal feed chains via raw material from fallen stock and dead animals (including also: ruminants, pigs, poultry, fish, wild/exotic/zoo animals, fur animals, cats, laboratory animals and fish) or via condemned meat. SSC, Opinion of the Scientific Steering Committee on the safety of ruminant blood with respect to TSE risks. SSC, Opinion of the Scientific Steering Committee on the risk of dissemination of brain particles into the blood and carcass when applying certain stunning methods. SSC, Opinion of the Scientific Steering Committee on the feeding of wild fishmeal to farmed fish and recycling of fish with regard to the risk of TSE. Taylor D.M., Fernie K., McConnell I., Steele P.J Observations on thermostable subpopulations of the unconventional agents that cause transmissible degenerative encephalopathies. Vet Microbiol. 64(1):33-8. Thackray, A. M., Ryder, S. J. and Bujdoso, R Modification of blood cell PrP epitope exposure during prion disease. Biochemical Journal, 390, The EFSA Journal (2007) 596, 19-45

20 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association Thackray, A. M., Fitzmaurice, T. J., Hopkins, L. and Bujdoso, R Ovine plasma prion protein levels show genotypic variation detected by C-terminal epitopes not exposed in cellsurface PrPC. Biochemical Journal, 400 (2): Wells, G. A., Hawkins, S. A. and Green, R. B Limited detection of sternal bone marrow infectivity in the clinical phase of experimental bovine spongiform (BSE): an update. Veterinary Record 144 (5): Wells, G., Spiropoulus, J., Hawkins, SAC, Ryder, SJ Pathogenesis of experimental bovine spongiform encephalopathy: Preclinical infectivity in tonsil and observations on the distribution of lingual tonsil in slaughtered cattle. The Veterinary Record (156): The EFSA Journal (2007) 596, 20-45

21 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association APPENDICES APPENDIX I: RESULTS OF THE CONTROL PROGRAMMES OF THE FEED BAN IN THE EU MEMBER STATES Percentage of infringements on the feed ban in the Member States (source EU Commission) Ruminant feed Non-ruminant feed Feed materials* N Samples % infr. N Samples % infr. N Samples % infr. Year % % % % % % % % % % % % % % % * Feed materials: various products of vegetable or animal origin, in their natural state, fresh or preserved, and products derived from the industrial processing thereof, and organic or inorganic substances, whether or not containing additives, which are intended for use in oral animal feeding either directly as such, or after processing, in the preparation of compound feedingstuffs or as carriers of premixtures. Article 2, Directive 96/25/EC(EC, 1996). EXPLANATORY NOTE TO THE RESULTS A significant number of the infringements were duplicates (from additional samples collected after 1 positive analysis) or rather infringements on labelling (fishmeal detected in nonruminant feed which is not prohibited but was not marked on the label). The real percentage of infringements may therefore be lower. Member States were also requested to target the controls which may have increased the percentage of infringements. Since the start of the extended feed ban (1 January 2001) in the European Union, the percentage of infringements continuously decreased. The slight increase in 2004 is mainly due to the amounts of remnants of meat-and-bone meal in production lines not systematically cleaned in the new Member States following Accession in May The results indicate that the number of infringements despite the very strict legal provisions at every stage (transport, storage, feed production, farms) does not become zero but remains at a very low level. This can be most likely explained by the adventitious presence of animal constituents in certain raw materials such as sugar beet pulp and fat and the presence of cadavers of rodents and birds in raw materials. REFERENCES FROM APPENDIX I EC Council Directive 96/25/EC of 29 April 1996 on the circulation of feed materials, amending Directives 70/524/EEC, 74/63/EEC, 82/471/EEC and 93/74/EEC and repealing Directive 77/101/EEC. Official Journal of the European Union 125: The EFSA Journal (2007) 596, 21-45

22 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association APPENDIX II: FARMED FISH PRODUCTION SYSTEMS AND THE FATE OF SEWAGE WATER 1. Farmed Fish Production Systems. European farmed fish hare produced in four principally different production systems. Most of marine ongrowing (largely salmonids and cod in Northwest Europe; seabream and seabass in the Mediterranean) is carried out in floating net-pens. Salmon is transferred to these pens at a size of g when they have become smolts, able to regulate their osmotic balance in saltwater. These open systems do not facilitate separation of faeces from the surrounding waters. Some net-pens are equipped with devices to trap uneaten feed after it has sunk through the bottom of the pens, and the trapped feed is normally directly re-fed to the fish. The production of salmon and rainbow trout smolts (from hatching until release into the sea) and juvenile marine fish is normally carried out in indoor tank facilities. These farms commonly employ sieves and filters to remove particle material from uneaten feed and faeces in order to minimise pollution from the farms. There is some inland production of freshwater fish in raceways. These systems permit some cleaning of the effluent water by filtration, but such cleaning is employed to a varying degree. There is some production in freshwater ponds, especially in east Europe. These semi-extensive systems have a much lower degree of water exchange than tanks and raceways. The ponds are normally harvested by draining the ponds and netting the fish, and sediments are normally utilised to stimulate primary production in the pond upon next filling with water. 2. Discharges of organic material and nitrogen from fish farms Figures concerning excretion of organic material and nitrogen are presented, while phosphorus is not presented since the sources, dietary concentrations, and availabilities of this nutrient varies so much from one production system and country to another. All examples are made based on production statistics and experiments with Atlantic salmon unless otherwise stated. (a) Freshwater stage The examples are calculated for Atlantic salmon growing from 0.15 to 100 g. The final whole-body composition per kg salmon ready for transfer to saltwater is 170 g crude protein (15.8 g N) and 80 g fat. An average macronutrient composition per kg feed used during this cycle is: water, 80 g; crude protein, 480 g (76.8 g N); fat, 250 g; starch, 80 g; indigestible carbohydrate, 50 g; Organic material, 860 g. The following apparent digestibility coefficients have been used based on Aslaksen et al. (2007), with a slight correction for more efficient starch digestion in freshwater than in saltwater (Storebakken, 2002): crude protein (and N), 0.85; fat, 0.90; starch, The example has been developed based on the assumption that 0.7 kg of this diet can provide 1 kg of body weight increase (feed conversion ratio (FCR)=0.7). This corresponds to a whole-body nitrogen retention of 50% of ingested nitrogen. Higher estimates (Storebakken et al., 1987a) have been obtained experimentally, indicating that this is may be a conservative estimate for loss of N in faeces. The EFSA Journal (2007) 596, 22-45

23 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association (b) Saltwater stage The examples are calculated for Atlantic salmon growing from 100 g to 4 kg. The composition of the growth per kg of salmon produced is 170 g crude protein (15.8 g N) and 140 g fat. An average macronutrient composition per kg feed used during this cycle is: water, 80 g; crude protein, 430 g (68.8 g N); fat, 350 g; starch, 80 g; indigestible carbohydrate, 50 g; organic material, 910 g. The following apparent digestibility coefficients have been used (average values based on Aslaksen et al., 2007): crude protein (and N), 0.85; fat, 0.90; starch, The example has been developed based on the assumption that 1.0 kg of this diet can provide 1 kg of body weight increase. This corresponds to a whole-body nitrogen retention of 40% of ingested nitrogen. In the example, an actual FCR at 1.15 kg fed per kg body weight increase was used. This was the realised overall FCR for the whole Norwegian salmon industry in the beginning of the 2000s (Storebakken, 2002). 3. The Fate of Sewage 3.1. Fate of Sewage in Freshwater Production Systems Figure 1 illustrates that the total burden of solids excreted from fish farms is highly dependent on the rate of overfeeding. The fish can obtain maximum growth virtually without overfeeding (Storebakken et al., 1987a,b). In this case solids discharge from the fish farm will mainly consist of faeces. Organic material waste 500 g organic material per kg produced FCR=0.7 FCR=1.0 FCR=1.2 Feed Fecal Figure 1. Estimated wastes of organic material per kg Atlantic salmon produced during the freshwater stage, from 0.15 to 100 g of fish weight. FCR= Food Conversion Ratio (kg fed per kg body weight increase). Effluent water from tank systems and raceways is normally cleaned by filters. Uneaten feed is very efficiently removed by simple filtration. The efficiency of removal of suspended particle material, mainly from faeces is, however variable, and depends both on the type of equipment used (Davidson and Sumerfelt, 2005), and optimal use of the filter in the aquacultural system (Brinker and Rösch, 2005). Recent examples illustrate that a microscreen filter drum can remove as much as 40-45% of the mass from outlet water of a fish farm (Davidson and Sumerfelt, 2005). Further treatment of the sludge from filtered water, for example with alum can effectively be The EFSA Journal (2007) 596, 23-45

24 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association used to remove and concentrate solids (82% of solids and 96% of total phosphorus) (Ebeling et al., 2006). As environmental regulations become more stringent, environmentally sound waste management systems and disposal practices are increasingly more important in all types of aquaculture (Ebeling et al., 2006). There is, however, no updated statistical information available on the use of modern sludge treatment equipment in European fish farming. However, if an optimal feeding technique is employed for production of salmon so that rate of overfeeding is less than 100 g per kg gain (it is very difficult to completely avoid overfeeding during the first part of the life of salmon), and that 90% of the uneaten feed is trapped by filters, as well as 40% of solids from faeces, the total amount of N released per kg of growth would be 26.2 g, whereof 71% originates from metabolic loss. Correspondingly, discharge of organic material would be 114 g, assuming that organic material is evenly distributed within the discarded solids. The metabolic loss of nitrogen is water soluble and not captured by conventional filtration and settling techniques. Efficient denitrification may, however, be efficiently carried out in the biological filters of recirculation systems. Not correcting for mortality and assuming that one smolt of 100 g is sufficient to produce a ready for slaughter salmon with a whole body weight at 4 kg, the release of nitrogen to the environment to the freshwater system in a well-managed farm is 655 g N per 1000 kg salmon produced in the sea, or 2.85 kg organic material. Thus, only a marginal part of the effluent produced through the total life cycle of a fish gaining most of the weight in open marine netpens can be controlled by filtration and sewage treatment Fate of Sewage in Floating Sea-Pens. The are currently no technologies available to remove faeces and metabolites from open, floating net-pens. The amount of uneaten feed discharged has been dramatically reduced over the last 20 years, and several farms produce fish at a feeding rate with virtually no feed wastage (Storebakken, 2002). Metabolic wastes have also been significantly reduced over the same timeperiod. Figures 2 gives an estimate of organic material discharges per kg produced in a well-managed Norwegian salmon farm, respectively. Even though both metabolic and faecal discharges may vary based on the composition of the feed, fish species, and environmental factors, the main key to reduced pollution from fish farms is the rate of feed loss (Åsgård et al., 1999). In this example, based on representative figures from salmon farming, the total discharge from the production of 1000 kg of salmon in the sea is 62.3 kg N, whereof 67% is metabolised by the fish and water soluble. The corresponding discharge of organic material is 321 kg. The EFSA Journal (2007) 596, 24-45

25 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association Organic material loss 200 g organic material per kg produced Fecal Feed Figure 2. Estimated wastes of organic material per kg Atlantic salmon from 100 g to 4 kg of fish weight. The salmon is produced in marine net-pens. Feed conversion ratio (FCR) is1.15 kg fed per kg body weight increase. REFERENCES FROM APPENDIX II Åsgård, T., Austreng, E., Holmefjord, I., Hillestad, M., Shearer, K., Resource efficiency in the production of various species. In Svennevig, N., Reinertsen, H., New, M. (Eds.) Sustainable Aquaculture. A.A. Balkema, Rotterdam, The Netherlands, pp Aslaksen, M.A., Kraugerud, O.F., Penn, M., Svihus, B., Denstadli, V., Jørgensen, H.Y., Hillestad, M., Krogdahl, Å., Storebakken, T., Screening of nutrient digestibilities and intestinal pathologies in Atlantic salmon, Salmo salar, fed diets with legumes, oilseeds, or cereals. Aquaculture, 272, Brinker, A., Rösch, R., Factors determining the size of suspended solids in a flow-through fish farm. Aquacult. Eng. 33, Davidson, J., Summerfelt, S.T., Solids removal from a coldwater recirculating system comparison of a whirl separator and a radial-flow settler. Aquacult. Eng. 33, Ebeling, J.M., Welsh, C.F., Rishel, K.L., Performance evaluation of an inclined belt filter using coagulation/flocculation aids for the removal of suspended solids and phosphorus from microscreen backwash effluent. Aquacult. Eng. 35, Storebakken, T., Austreng, E., 1987a. Ration level for salmonids. I. Growth, survival, body composition, and feed conversion in Atlantic salmon fry and fingerlings. Aquaculture 60, Storebakken, T., Austreng, E., 1987b. Ration level for salmonids. II. Growth, feed intake, protein digestibility, body composition, and feed conversion in rainbow trout weighing kg. Aquaculture, 60: Storebakken, T., Chapter 6. Atlantic salmon, Salmo salar. In Webster, C. and Lim, C. (Editors). Nutrient Requirements and feeding of Aquaculture Fish. CAB International Publishers. The EFSA Journal (2007) 596, 25-45

26 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association APPENDIX III: SUMMARY OF RESEARCH FINDINGS ON TSE INFECTIVITY IN BLOOD OF ANIMALS WITH TSE When making an interpretation of the data collected in this table, a number of factors have to be taken into account with regard to: Number of animals and species involved (i.e. species barrier, also considering transgenic mice nature); Amount of inoculum and infectious titre; Assay sensitivity for PrPSc and/or infectivity detection in the receptor. Further details can be found in the references given, but were not included in this report as the Working Group considered that they would not affect the outcome of this risk assessment when answering the terms of reference. Species Route of Pos./total Donor Assay Inoculum 5 Inoculation donors Reference BSE (natural) Cow Mouse Blood clot/serum/ ic + ip 0/2 Fraser, 1994, cited Buffy coat ic + ip 0/2 in Bradley 1999 BSE (experimental) Cow Mouse Buffy coat ic + ip 0/11 (pools) Wells, 2005 Cow Buffy coat ic 0/4 (pools) Wells, 2005 Mouse Mouse Plasma ic 4/48 Taylor, 2000 Sheep Sheep Whole blood iv(transfusion) 1/19 Houston, 2000 Microcebe Microcebe Buffy coat ic 1/1 Bons, 2002 Cow Cow Buffy coat ic 0/4 (pools) Wells, 2005 Monkey Monkey Buffy coat iv 1/7 Lasmezas, 2005 Whole blood iv 0/2 Lasmezas, 2005 Buffy coat ic 0/1 Lasmezas, 2005 Plasma ic 0/1 Lasmezas, 2005 Cow Mouse Whole blood ic 0/5 Espinosa, 2007 Sheep Mouse Buffy coat ic+ip 0/4 Bellworthy, 2005 Scrapie (natural) Goat Mouse Blood clot/serum ic 0/3 Hadlow, 1980 Sheep Mouse Blood clot/serum ic 0/18 Hadlow, 1982 Sheep Sheep Whole blood iv 4/10 Hunter, In several of the studies, assays were conducted on serial specimens obtained during both the incubation and clinical phases of disease. The EFSA Journal (2007) 596, 26-45

27 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association Species Route of Pos./total Donor Assay Inoculum 6 Inoculation donors Reference Scrapie (experimental) Goat Goat Whole blood ic 0/14 Pattison, 1962 Mouse Mouse Whole blood ic 0/39 Eklund, 1967 Goat Mouse Blood clot ic or sc 0/20 Hadlow, 1974 Sheep Mouse Serum ic 1/1 Gibbs, 1965 Rat Rat Serum ic 1/1 (pool) Clarke, 1967 Mouse Mouse Serum ic 1/1 (pool) Clarke, 1967 Mouse Mouse Whole blood ic 3/13 Dickinson, 1969 Hamster Hamster Whole blood ic 0/9 Diringer, 1984 Hamster Hamster Blood extract ic 5/5 (pools) Diringer, 1984 Mouse Mouse Whole blood ic 3/13 Dickinson, 1969 Hamster Hamster Whole blood ic 0/9 Diringer, 1984 Hamster Hamster Blood extract ic 5/5 (pools) Diringer, 1984 Hamster Hamster Blood extract ic 10/11 (pools) Casaccia, 1989 Hamster Hamster All blood components ic 1/1 (large pool) Rohwer, 1999 Hamster Hamster Whole blood ic 31/124 Rohwer, 2002 Whole blood iv 3/112 Rohwer, 2002 Whole blood ic 31/124 Rohwer, 2002 Whole blood iv 3/112 Rohwer, 2002 Mink encephalopathy (experimental) Mink Mink Serum ic 0/2 Marsh, 1969 Mink Mink Whole blood, plasma, ic 0/8 (pools) Marsh, 1973 red cells, white cells, platelets scjd (experimental) Guinea pig Guinea pig Buffy coat ic,sc,im,ip 10/28 Manuelidis, 1978 Monkey Monkey Whole blood iv 0/4 Brown, 2004 Chimpanzee Monkey Leucocytes ic+iv 0/2 Brown, 2004 Plasma ic+iv 0/2 Brown, 2004 Platelets ic+iv 0/3 Brown, 2004 Red cells ic+iv 0/3 Brown, In several of the studies, assays were conducted on serial specimens obtained during both the incubation and clinical phases of disease. The EFSA Journal (2007) 596, 27-45

28 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association vcjd (experimental) Mouse Mouse Buffy coat ic+iv 4/4(pools) Gervenakova, 2003 Plasma ic+iv 4/4(pools) Gervenakova, 2003 Monkey Monkey Whole blood iv 0/4 Brown, 2004 Buffy coat ic 0/2 Brown, 2004 GSS (experimental) Mouse Mouse Buffy coat ip 4/7 (pools) Kuroda, 1983 Mouse Mouse Buffy coat/plasma ic 5/5 (pools) Brown, 1999 Buffy coat/plasma iv 2/2 (pools) Brown, 1999 Chimpanzee Monkey Leukocytes ic+iv 1/1 Brown, 2004 Plasma ic+iv 0/1 Brown, 2004 Platelets ic+iv 0/1 Brown, 2004 Red cells ic+iv 0/1 Brown, 2004 REFERENCES FROM APPENDIX III Bellworthy, S. J., Hawkins, S. A., Green, R. B., Blamire, I., Dexter, G., Dexter, I., Lockey, R., Jeffrey, M., Ryder, S., Berthelin-Baker, C. and Simmons, M. M Tissue distribution of bovine spongiform encephalopathy infectivity in Romney sheep up to the onset of clinical disease after oral challenge. Veterinary Record 156 (7): Bons N, Lehmann S, Mestre-Frances N, Brain and buffy coat transmission of bovine spongiform encephalopathy to the primate Microcebus murinus. Transfusion, 42, Bradley, R BSE transmission studies with particular reference to blood. Developments in Biological Standardisation, 99, Brown, P., Cervenakova, L., McShane, L.M., Further studies of blood infectivity in an experimental model of transmissible spongiform encephalopathy, with an explanation of why blood products do not transmit Creutzfeldt-Jakob disease in humans. Transfusion, 39, Brown, P Up date on Baxter monkey TSE research. Presented at the 8 th annual Cambridge Healthtech Institute Conference on Transmissible Spongiform Encephalopathies, Washington, DC, February 23-24, Casaccia, P., Ladogana, A., Xi, Y.G., Pocchiari, M Levels of infectivity in the blood through out the incubation period of hamsters peripherally injected with scrapie. Archives of Virology, 108, Cervenakova, L., Yakovleva, O., McKenzie, C., Similar levels of infectivity in the blood of mice infected with human derived vcjd and GSS strains of transmissible spongiform encephalopathy. Transfusion, 43, Clarke, M.C., Haig, D.A Presence of the transmissible agent of scrapie in the serum of affected mice and rats. Veterinary Record, 80, 504. The EFSA Journal (2007) 596, 28-45

29 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association Dickinson A.B., Meikle, V.M.H., Genetic control of the concentration of ME7 scrapie agent in the brain of mice. Journal of Comparative Pathology, 79, Diringer, H Sustained viremia in experimental hamster scrapie. Archives of Virology, 82, Eklund, C. M., Kennedy, R. C. and Hadlow, W. J Pathogenesis of scrapie virus infection in the mouse. Journal of Infectious Diseases, 117 (1): Espinosa, J.C., Morales, M., Castilla, J., Rogers, M. and Torres, J. M Progression of prion infectivity in asymptomatic cattle after oral bovine spongiform encephalopathy challenge. Journal of General Virology, 88, ( pt.4), Gibbs Jr., C. J., Gajdusek, D. C., Morris A Viral characteristics of the scrapie agent in mice. In: Gajdusek DC, Gibbs CJ Jr, Alpers M, eds. Slow, latent, and temperate virus infections. NINDB Monograph no. 2. PHS Publication no Washington, DC: US Government Printing Office, 1965, Hadlow, W. J., Eklund, C. M., Kennedy, R. C., Jackson, T. A., Whitford, H. W. and Boyle, C. C Course of experimental scrapie virus infection in the goat. Journal of Infectious Diseases, 129 (5): Hadlow, W. J., Kennedy, R. C., Race, R. E. and Eklund, C. M Virologic and neurohistologic findings in dairy goats affected with natural scrapie. Veterinary Pathology 17 (2): Hadlow, W. J., Kennedy, R. C. and Race, R. E Natural infection of Suffolk sheep with scrapie virus. Journal of Infectious Diseases, 146 (5): Houston, F., Foster, J., Chong, A., Hunter, N. and Bostock, C Transmission of BSE by blood transfusion in sheep. The Lancet. 356, Hunter, N., Foster, J., Chong, A., McCutcheon, S., Parnham, D., Eaton, S., MacKenzie, C. and Houston, F Transmission of prion diseases by blood transfusion. Journal of General Virology, 83, (Pt 11), Kuroda, Y., Gibbs, C.J. Jr, Amyx, H., Creutzfeldt-Jakob disease in mice: Persistent viremia and preferential replication of virus in low density lymphocytes. Infecttion and Immunology, 41, Lasmézas, C.I Pathogenesis of vcjd in non-human primates. Presented at the 9th Annual Cambridge Healthtech Institute Conference on Transmissible Spongiform Encephalopathies, McLean, VA, February Manuelidis, E.E., Gorgacz,E.J., Manuelidis, L., Viremia in experimental Creutzfeldt-Jakob disease. Science, 200, Marsh R.F., Burger D., Hanson R.P Transmissible mink encephalopathy: Behavior of the disease agent in mink. American Journal of Veterinary Research, 30, Marsh, R.F., Miller, J.M., Hanson, R.P Transmissible mink encephalopathy: Studies on the peripheral lymphocyte. Infection and Immunology 7, Pattison, I.H., Millson G.C Distribution of the scrapie agent in the tissue of experimentally inoculated goats. Journal of Comparative Pathology,(72): The EFSA Journal (2007) 596, 29-45

30 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association Rohwer, R.G New data on blood-borne TSE infectivity. Presented at the 6th Annual Cambridge Healthtech Institute Conference on Transmissible Spongiform Encephalopathies, Washing ton, DC, February 6-7, Taylor, D.M., Fernie, K., Reichel, H.E., Somerville, R.A Infectivity in the blood of mice with a BSE-de rived agent (letter). Journal of Hosp Infections, 46, Wells, G.A.H., Spiropoulos, J., Hawkins, S.A.C., Ryder, S.J Pathogenesis of Experimental bovine spongiform encephalopathy: Preclinical infectivity in tonsil and observations on the distribution of lingual tonsil in slaughtered cattle. Veterinary Record, 156, The EFSA Journal (2007) 596, 30-45

31 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association APPENDIX IV: THE DIFFERENT STAGES OF THE CATTLE SLAUGHTER PROCESS (1) Pre-slaughter handling and transfer of cattle into the stunning area/pen Slaughter cattle are normally moved from a lairage pen through a race into a stunning box where restraint is achieved prior to stunning and slaughter. There are conventional more simple pens as well as those that employ various restraining devices such as belly plate, rump push and neck restraint devices. (2) Restraint Restraint is essential for correct stunning application so that optimum target is hit. In the case of stunning with a captive bolt the intersection point of lines drawn between the eyes and horns/ buds needs to be aimed at requiring neck restraint. Electrical stunning is usually carried out using an automated system comprising a special pen and electrical stunning tongs that are applied automatically. For religious slaughter (e.g. Shechita) chin lifts and rotating pens can also be used. (3) Stunning or neck cutting (only Shechita and some Halal slaughter) Stunning should result in immediate collapse of the animal in the pen. In case of religious slaughter without stunning the neck cut can be carried out whilst the animals is in the pen. (4) Removal from pen Following stunning the collapsed of animal is removed using the revolving side of the pen usually onto a cradle outside the pen after restraint devices are released. (5) Shackling by a hind leg Whilst the animal is on the cradle a shackle is applied to one of the hind legs and attached to an elevating hoist line. (6) Hoisting Hoisting is the usual method that suspend the animal in the vertical position before exsanguination. However, some systems use conveying moving tables where animals are moved in horizontal position. (7) Exsanguination (sticking) by a neck or chest cut This procedure (sticking) involves either a transverse neck cut or a chest cut into the thoracic cavity to severe vessels near the heart. Blood collection during exsanguination may be carried out following two different practices: Employing the Hollow Knife system, where blood gushes from the stick hole directly through a plastic tube incorporated in the knife into a tank. Allowing blood to loosely drop into a canal or collection through on the floor of the bleeding area. Blood from different animals is usually pooled at the collection point. The EFSA Journal (2007) 596, 31-45

32 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association (8) Dehiding and evisceration Following the bleeding out, animals are dehided and eviscerated by removing the internal organs. Normally, the sequence of the individual operations leading to the final harvesting of a beef carcase occurs in a production line. This follows a continuous flow with separation both in time and space. Thus, the area where blood is collected falls distant from that where the carcase is longitudinally split through the vertebral column. However and in a limited number of cases, small cattle abattoirs may carry out the full dressing operation in a small room while the carcase is hanging still immediately after exsanguination. A small area is designated for the previous bleeding out of the carcase, but within the proximity to the place where the rest of the operations are carried out including splitting. The EFSA Journal (2007) 596, 32-45

33 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association APPENDIX V: STUNNING METHODS AND THEIR RELATIVE EFFECTS ON BRAIN TISSUE EMBOLISM 1. Mechanical stunning methods (a) Penetrating Captive Bolt method (PCB). This is the most commonly used stunning method for cattle in the EU. Sheep can also be stunned by this method. The method involves a steel bolt fired from a gun powered by a blank cartridge, or alternatively, by compressed air. The bolt (length mm; diameter mm) extends from the gun for up to its entire length and penetrates the skull but remains attached to the gun. The bolt must be aimed at the forehead point i.e. the cross-over of imaginary lines drawn between the base of the horns and the contra lateral eyes and no further away than 2 cm radius from this point (see figure 1 in the annex to the EFSA Opinion on BSE risk from dissemination of brain particles in blood and carcass following stunning (EFSA, 2004)). Previous research indicated that the impact of the bolt with the cranium is the principle determinant of effective stunning, rather than the penetration of the bolt into the brain tissue (Daly and Whittington, 1989). If used correctly, penetrating captive bolt stunning can be 100% effective in adult cattle. However, inadequate stunning can occur due to insufficient head restraint, wrong position of the operator and/or inadequate quality/maintenance of the gun. Bleeding is performed immediately after the shot, which also ensures the death of the animal. Potential public health concerns from TSE infected animals have been considered and reviewed (Anil and Austin, 2003). In cattle stunned with a penetrating captive bolt (PCB) method a frequency of 4% CNS embolism in jugular blood has been reported (Coore et al., 2004ab; Coore et al., 2005). In sheep, higher frequencies (23% and 14% respectively for cartridge activated and pneumatically activated guns) of CNS embolism in jugular blood have been reported (Anil and Harbour, 2001; Coore et al., 2004ab). As the heart continues pumping for several minutes between the stunning and the end of exsanguinations, some of the embolic CNS material dislodged by the penetrating captive bolt gun might enter venous blood vessels draining the head and consequently be disseminated to other organs/tissues. This can happen not only with use of a penetrating gun that injects air into the brain (Schmidt et al., 1999) but also when stunning is performed without air injection (Anil et al., 2002; Coore et al., 2004ab; Coore et al., 2005). If the CNS emboli contained the BSE agent, the potential public health risks could be associated with internal, haematogenous contamination of edible tissues/organs. The total amount of CNS material (and thus its level of potential infectivity) released and entering the venous vessels (e.g. sagittal sinus) in during captive bolt stunning of slaughtered animals is difficult to determine and probably varies. An average amount of (SE) g of loose brain material collected from animals (n=20) stunned with a PCB gun has been reported, however, this could be as high as 10 g (Anil et al., 2002). To contaminate the carcass meat and the visceral organs, CNS emboli carried from the brain by venous blood via the right heart to the lungs would have to pass through the lungs capillary system and, via the left heart, reach aorta i.e. the systemic arterial circulation. Whether the CNS material would pass through the lungs capillary system or not, is determined primarily by the ratio between the size of the emboli and the diameter of small vessels. Recent research showed The EFSA Journal (2007) 596, 33-45

34 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association that CNS material experimentally injected in jugular vein could be detected in blood collected from the aorta from sheep (Coore et al., 2004ab). In addition to haematogenous contamination of edible tissues with CNS material, other public health concerns may also be associated with PCB methods. For example, cross- or airborne contamination of the stunning gun operator, the environment such as the stun-box and/or the animals consecutively stunned with the same gun could occur, based on studies using experimental contamination with marker bacteria (Daly and Whittington, 1989; Buncic et al., 2002; Prendergast et al., 2004). Furthermore, the hide of the animals stunned with a PCB, particularly on the frontal area of the head can be contaminated by leakage of CNS material from the stun hole. This was reported from commercial abattoirs in France and whilst such a risk can be reduced by use of a plug to seal the stun hole (EC 2001), this practice is only compulsory when harvesting cheek meat and only under certain prescribed circumstances. Furthermore, such a leakage from the stun hole could lead to contamination of the abattoir waste water and the wider environment since grids of abattoir drains trap only material larger than 6 mm (EC, 2002) which is treated as Specified Risk Material (SRM). (b) Non-Penetrating (percussion) Captive Bolt method (NPCB). Non-penetrating captive bolt (NPCB) method is used for stunning cattle, but less commonly than the penetrating captive bolt (PCB) method. The non-penetrating device operates on the same principle as the penetrating captive bolt gun (described above), but the tip of bolt is mushroom-shaped and should deliver a percussive blow to the head without penetrating the skull. Potential public health concerns have already been considered. Recent reports indicated that CNS embolism can be present in mechanically stunned animals even when non-penetrating CB method is used. In NPCB stunned cattle, CNS material was detected in jugular blood of 2% animals (Coore et al., 2004ab; Coore et al., 2005). The reasons for the embolism primarily relate to the fact that, as mentioned above, the blow can cause extensive damage of the brain tissue even without the skull penetration, but also the blow can cause inward skull fracture with the broken bone penetrating the brain. 2. Electrical stunning Electrical stunning is used infrequently for cattle in the EU apart from some individual member states (e.g. Germany, UK and Ireland), but in some other countries e.g. New Zealand it is used regularly. Electrical stunning involves application of a sufficient electric discharge through the brain of a restrained animal and causes an epileptiform insult and immediate loss of consciousness. Two main electrical stunning techniques are used for cattle: head-only stunning (positions eye-eye or eye-ear), and head-to-body stunning (also called electrical stun/kill method). Head-only electrical technique stunning can be based on use of various manual or automatic stunners. Major drawbacks of head-only technique in cattle/calves are short duration of the epileptiform insult and the occurrence of clonic convulsions. The former can be resolved by induction of cardiac ventricular fibrillation or rapid sticking (e.g. chest sticking is advised). The latter can be stopped by electro-immobilisation a low voltage, frontto-rear (spinal) discharge applied before sticking. Head-to-body stunning (i.e. stun/kill) technique can be performed on restrained cattle in a box by a split application of an electric current in two stages: the current is first applied to the head by tongs, followed by an application of the tongs across the chest in a position that enables the current to pass through The EFSA Journal (2007) 596, 34-45

35 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association the heart. In the first stage, the applied current should be sufficient to cause unconsciousness, whilst the low frequency current used in the second stage should cause ventricular fibrillation, cardiac arrest and death. For sheep the head-only technique is used, with electrodes placed between the eyes and the base of the ears on both sides, that can be carried out either individually within the group (in pen) or on individual animals in a restrainer. Head-to-back (stun/kill) technique involves passing a current simultaneously through the brain and through the heart of the animal held in a restrainer. The electrical methods are generally perceived as more effective and suitable for calves and small ruminants than for adult cattle. There is no published evidence indicating that CNS emboli can be generated with electrical stunning methods and therefore the public health concerns for spread of BSE associated with this method are non-existing at present. 3. Gas stunning Gas stunning, used in pigs and poultry, is not suitable for ruminants at present due mainly to practical reasons (e.g. large animals, absorbent wool and high costs). Novel and potentially pain-free methods are also under investigation and they may offer good alternatives such as magnetic stunning (Anil et al., 2000). Consideration should also be taken to those cases where slaughtering of cattle is carried out without stunning. With some religious methods of slaughter such as Jewish/Shechita or Muslim/Halal 7 (Anil and Sheard, 1994; Anil et al., 2004; Rosen, 2004), the animal is usually restrained and slaughtered without stunning, by cutting the underside of the neck using a very sharp knife (FAO, 2004). There are no obvious/conceivable risks of dissemination of CNS embolism associated with neck cutting without stunning. 7 Religious slaughter methods which are usually carried out by bleeding the animals with a particular procedure without previous stunning. Thus, the risk of CNS embolism due to stunning is inexistent. The EFSA Journal (2007) 596, 35-45

36 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association A table summarising findings in this annex is included in Table 1. Table 1. Summary of brain tissue embolism findings related to the different stunning methods. It also provides a view on alternative approaches. Stunning method Species used in Brain tissue embolism Remarks Penetrating captive bolt cartridge activated Penetrating captive bolt with air injection Penetrating captive bolt with pithing Penetrating captive bolt pneumatically activated Non-Penetrating captive bolt cartridge activated Cattle, sheep Yes, (incidence: 4 %) Cattle Cattle Sheep Cattle Cattle Sheep (Coore et al., 2004ab, 2005). Yes Anil et al., 1999 Yes Anil et al., 1999 Yes 14% Coore et al., 2004ab Anil et al, 1999 Coore et al. 2004ab, 2005 Not tested but likely Yes (Incidence 2%) Yes (Incidence 23%) Coore et al. 2004ab,2005 Electrical stunning Cattle, sheep No positive evidence, Highly unlikely Electrical stunning, novel stunning may offer an alternative. Banned in Banned in Novel methods may offer an alternative. Modifications to existing design as an alternative. Modifications to existing design as an alternative. Potential future novel methods to be developed Offers an alternative to captive bolt stunning methods. Gas stunning Pigs, poultry Highly unlikely Unsuitable, impracticable for ruminants No stunning (Religious slaughter, only neck cut) Ruminants, poultry Highly unlikely Animal Welfare objections The EFSA Journal (2007) 596, 36-45

37 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association REFERENCES FROM APPENDIX V Anil, H. and Sheard, P Welfare aspects of religious slaughter. Meat Focus International (October): Anil, M. H., Butler, S. R., Johnson, C. B. and McKinstry, J. L Suprression of somatosensory evoked potentials by transcranial magnetic stimulation in the rabbit. Proceedings of the Physiological Society: 526P. Anil, H. and Harbour, D. A Current stunning and slaughter methods in cattle and sheep: potential for carcass contaminatinon with central nervous tissue and microorganisms. Fleischwirtschaft International (3): Anil, H., Krailadsiri, P. and Seghatchian, J Changes in haematological parameters following captive bolt stunning in relation to the increased level of Syntaxin 1-B, a CNS marker in blood. Transfus Apher Sci 26 (2): Anil, H. and Austin, A Bovine spongiform encephalopathy: a review of some factors that influence meat safety. Livestock and Animal Feed, AGRIPPA. Available at Anil, M. H, Yesildere,T., Aksu, H., Matur, E., McKinstry, J. L., Erdogan, O., Hughe, S., Mason, C Comparison of religious slaughter of sheep with methods that include preslaughter stunning and the lack of differences in exanguination, packed cell volume and quality parameters. Animal Welfare 4 (13): Buncic, S., McKinstry, J. L., Reid, C. A. and Anil, M. H Spread of microbial contamination associated with penetrative captive bolt stunning of food animals. Food control (13): Coore, R. R., Barr, F. J., McKinstry, J. L. and Anil, M. H. 2004a. Neural embolism and cerebral venous drainage at stunning and slaughter. Vet Rec 155 (3): Coore, R. R., Love, S., McKinstry, J. L., Weaver, H. R., Phillips, A., Hillman, T., Hiles, M. J., Shand, A., Helps, C. R. and Anil, M. H. 2004b. Dissemination of brain emboli following captive bolt stunning of sheep: capacity for entry into the systemic arterial circulation. J Food Prot 67 (5): Coore, R. R., Love, S., McKinstry, J. L., Weaver, H. R., Philips, A., Hillman, T., Hiles, M., Helps, C. R. and Anil, M. H Brain tissue fragments in jugular vein blood of cattle stunned by use of penetrating or nonpenetrating captive bolt guns. J Food Prot 68 (4): Daly, C. C. and Whittington, P. E Investigation into the principal determinants of effective captive bolt stunning of sheep. Res Vet Sci 46 (3): EC, Regulation (EC) No 999/2001 of the European Parliament and of the Council of 22 May 2001 laying down rules for the prevention, control and eradication of certain transmissible spongiform encephalopathies. Official Journal of the European Union 147: EC, Regulation (EC) No 1774/2002 of the European Parliament and of the Council of 3 October 2002 laying down health rules concerning animal by-products not intended for human consumption Journal Volume (Issue): 1-95 EFSA, Opinion on BSE risk from dissemination of brain particles in blood and carcass following stunning. The EFSA Journal (123): The EFSA Journal (2007) 596, 37-45

38 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association FAO Anil, M.H. and Fisher, A.V. (Eds) A Manual of Good Practices for the Meat INdustry. Published by the Food and Agriculture Organisation of the United Nations and Carrefour.: Prendergast, D. M., Sheridan, J. J., Daly, D. J., McDowell, D. A. and Blair, I. S The use of a marked strain of Pseudomonas fluorescens to model the spread of brain tissue to the musculature of cattle after shooting with a captive bolt gun. J Appl Microbiol 96 (3): Rosen, S. D Physiological insights into sechita. Veterinary Record (154): Schmidt, G. R., Hossner, K. L., Yemm, R. S. and Gould, D. H Potential for disruption of central nervous system tissue in beef cattle by different types of captive bolt stunners. J Food Prot 62 (4): The EFSA Journal (2007) 596, 38-45

39 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association APPENDIX VI: TESTS TO IDENTIFY THE PRESENCE OF CENTRAL NERVOUS SYSTEM TISSUE IN BLOOD. (a) Glial Fibrillary Acidic Protein (GFAP) ELISA. Most of the described techniques have been mainly designed for detection of GFAP as a CNS-marker in meat (Schmidt et al., 1999; Schmidt et al., 2001; Schmidt et al., 2002). It was shown that, among these tests, a colorimetric microtitre plate-based sandwich GFAP ELISA is applicable to bovine blood (Schmidt et al., 1999). Its limit of detection is about 1 ng of GFAP and in dose response experiments it was established that the linear range extended up to 40 ng of GFAP (Schmidt et al., 1999) and that 0.2 to 0.8 µg of brain protein were within this range. GFAP ELISA has also been employed for detecting brain tissue in cattle and sheep blood reliably in recent years (Coore et al., 2004ab). A chemiluminometric GFAP ELISA specifically designed for use on serum sample has been described more recently (Vissers et al., 2006). It uses polyclonal rabbit anti-cow GFAP antibodies and has a limit of detection of 14 ng/l of GFAP in serum (and as 100 µl of serum was used per well in the test, it means that the LOD is about 1.4 pg of GFAP). (b) Syntaxin 1B ELISA. This method targets another suitable CNS-marker in blood and has a detection limit of 2-4 µg of brain protein/ml corresponding to approximately µg/ml of brain (Love et al., 2000). It is applicable on whole blood samples (Love et al., 2000; Anil et al., 2002). (c) NSE (neural enolase 2) and S100 ELISA. These two proteins appear as less suitable CNS-markers (Love et al., 2000; Vissers et al., 2006; Villmann et al., 2007) and are therefore not considered. A new possible marker, myelin proteolipid protein (PLP), has been suggested and has the advantage of being sensitive as well as heat-resistant (Villmann et al., 2007). However, its performance of the immuno-assay based on this marker has not yet been tested in blood. The EFSA Journal (2007) 596, 39-45

40 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association REFERENCES FROM APPENDIX VI Anil, H., Krailadsiri, P. and Seghatchian, J Changes in haematological parameters following captive bolt stunning in relation to the increased level of Syntaxin 1-B, a CNS marker in blood. Transfus Apher Sci 26 (2): Coore, R. R., Barr, F. J., McKinstry, J. L. and Anil, M. H. 2004a. Neural embolism and cerebral venous drainage at stunning and slaughter. Vet Rec 155 (3): Coore, R. R., Love, S., McKinstry, J. L., Weaver, H. R., Phillips, A., Hillman, T., Hiles, M. J., Shand, A., Helps, C. R. and Anil, M. H. 2004b. Dissemination of brain emboli following captive bolt stunning of sheep: capacity for entry into the systemic arterial circulation. J Food Prot 67 (5): Love, S., Helps, C. R., Williams, S., Shand, A., McKinstry, J. L., Brown, S. N., Harbour, D. A. and Anil, M. H Methods for detection of haematogenous dissemination of brain tissue after stunning of cattle with captive bolt guns. J Neurosci Methods 99 (1-2): Schmidt, G. R., Hossner, K. L., Yemm, R. S., Gould, D. H. and O'Callaghan, J. P An enzyme-linked immunosorbent assay for glial fibrillary acidic protein as an indicator of the presence of brain or spinal cord in meat. J Food Prot 62 (4): Schmidt, G. R., Yemm, R. S., Childs, K. D., O'Callaghan, J. P. and Hossner, K. L The detection of central nervous system tissue on beef carcasses and in comminuted beef. J Food Prot 64 (12): Schmidt, G. R., R.S., Y., K.D., C., J.P., O. and K.L., H Verification of different glial fibrillary acidic protein (GFAP) analyses as accurate detectors of central nervous system tissue in advanced meat recovery (AMR) products. Meat Science (62): Villmann, C., Sandmeier, B., Seeber, S., Hannappel, E., Pischetstrieder, M. and Becker, C. M Myelin proteolipid protein (PLP) as a marker antigen of central nervous system contaminations for routine food control. Journal of Agricultural and Food Chemistry 17 (55): Vissers, J. L., Mersch, M. E., Rosmalen, C. F., van Heumen, M. J., van Geel, W. J., Lamers, K. J., Rosmalen, F. M., Swinkels, L. M., Thomsen, J. and Herrmann, M Rapid immunoassay for the determination of glial fibrillary acidic protein (GFAP) in serum. Clin Chim Acta 366 (1-2): The EFSA Journal (2007) 596, 40-45

41 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association APPENDIX VII: TESTS TO IDENTIFY THE PRESENCE OF BOVINE BLOOD CELLS AND THEIR LIMITATIONS IN FISH FEED. (a) Microscopy The use of classical microscopy, the reference method for detection of animal constituents in feed (EC, 2003) enables, at least up to a certain point, detection of spray dried blood products in feed. The cellular blood fraction will often give dark fragments mostly fine spherical particles of irregular shape, hard to break, with a smooth surface but dull or lacking luster, according to Sanches (Sanches et al., 2006), and their blood nature can be tested chemically in two ways (Vary et al., 2007): i) the iron present in haemoglobin should react with 3, 3, 5, 5 - tetramethylbenzidine to produce a blue/green colour and ii) the catalase of blood can be evidenced by use of hydrogen peroxide and this should give rise to visible air bubbles corresponding to the formation of oxygen. However, these tests which will never determine the species of origin are aimed in feed that is made of a vegetal matrix. How will they work in presence of fish meal that normally also contains fish blood is not known. Moreover, the sensitivity of this method is not sufficiently explored. As an indication, it is worthwhile to cite that according to Sanches et al. (2006), the simple visual recognition of the blood particles has a LOD below 0.1%. It is merely an indication because the different solvents considered for sedimentation in the methods used by the latter authors don t match with the one given in the official method (EC, 2003). Finally, in fish feed, spray dried blood cells as ingredient are often completely haemolysed making it much more difficult to distinguish the structures on which microscopists generally rely to identify blood. As a consequence, it appears that, for determination of blood products in fish feed, microscopy is of rather low value but it remains useful for detection of prohibited processed animal proteins in fish feed. (b) Polymerase Chain Reaction Polymerase chain reaction (PCR) is a possible identification method to determine the animal origin of spray dried blood products (Castello et al., 2004), even on spray dried red cells that in fact do not contain only erythrocytes but also other blood cells. The technique should therefore also work directly on feed because the degree of difficulty to find species-specific DNA-targets on such an ingredient in feed is certainly not higher than for detection of rendered animal material in feed. Nevertheless, PCR can only assign a species origin to the detected DNA, it is unable to make a link with the kind of tissue from which the product originated. This means that bovine spray dried blood (or red) cells could give the same PCR signal as unauthorized bovine processed animal proteins (PAPs). That is why PCR as a detection method is not sufficient in this case to guarantee that the bovine DNA targets found are solely coming from bovine spray dried blood cells as a source of animal proteins. Use of bovine spray-dried blood cells in fish feed, if authorized, therefore introduces at analytical level a higher risk of confusion between presence of authorized and unauthorized bovine products in this kind of feed. The EFSA Journal (2007) 596, 41-45

42 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association (c) Immunoassays Immunoassays targeting species-specific proteins (or in fact more precisely species-specific parts of such proteins) were described to detect presence of bovine blood in porcine plasma or whole blood (Newgard et al., 2002; Polo et al., 2004) but these techniques were only valid on the blood product not in feed. More recently, specific immunoassays targeting thermostable bovine blood serum proteins applicable to feed have been reported (Hsieh et al., 2007). These tests should be validated in the fish feed containing bovine SDRC because they target serum proteins while bovine spray dried blood cells consist of the cellular blood fraction not the plasma fraction. Even if it is very likely that some of these thermostable blood serum proteins are left in the cellular blood fraction, the resulting sensitivity of the test on blood products in feed has to be checked (no LOD is yet reported for these immunoassays). (d) Other tests Discrimination power of near infrared microspectrometry (which determines spectral characteristics of the particle in the IR-spectrum) towards blood particles (be it from bovine SDRC or not) within other feed ingredient particles is unknown. REFERENCES FROM APPENDIX VII Castello, A., Francino, O., Cabrera, B., Polo, J., Sanchez, A Identification of bovine material in porcine spray-dried blood derivatives using the polymerase chain reaction technique. Biotechnology Agronomy Society and Environment 4 (8): EC, Commission Directive 2003/126/EC of 23 December 2003 on the analytical method for the determination of constituents of animal origin for the official control of feedingstuffs. Journal Volume (Issue): Hsieh, Y. H., Ofori, J. A., Rao, Q. and Bridgeman, C. R Monoclonal antibodies specific to thermostable proteins in animal blood. J Agric Food Chem 55 (16): Newgard, J. R., Rouse, G. C. and McVicker, J. K A novel method for detecting bovine immunoglobulin G in dried porcine plasma as an indicator of bovine plasma contamination. Journal of Agricultural and Food Chemistry 11 (50): Polo, J., Saborido, N., Rodenas, J. and Rodriguez, C Determination of the presence of bovine immunoglobulin G in liquid or spray-dried porcine and whole blood by agar gel immunofiffusion. Journal of AOAC International 1 (87): Sanches, R. L., Alkmin-Filho, J. F., de Souza, S. V. C. and Junqueira, R. G In-house validation of a method for detection of animal meals in ruminant feeds by microscopy. Food Control (17): Vary, N., Makowski, J. and van Raaamsdonk, L Focusing on detecting and identifying ingredients of animal origin and the American/Canadian ruminant feed bans. Intermediate/advanced agricultural short course May 2007, Laval University Quebec, Canada, AOCS Feed microscopy division.: The EFSA Journal (2007) 596, 42-45

43 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association GLOSSARY / ABBREVIATIONS Term Animal By- Products Definition Entire bodies or parts of animals or products of animal origin referred to in Articles 4, 5 and 6 (of the Reg. CE 1774/2002) not intended for human consumption, including ova, embryos and semen. Blood Products As defined in Regulation (EC) 1774/2002 of the 3 rd October 2002: Products derived from blood or fractions of blood, excluding blood meal; they include dried/frozen/liquid plasma, dried whole blood, dried/frozen/liquid red cells or fractions thereof and mixtures. Blood employing for the manufacturing of blood products has to be sourced from animals that have passed both ante mortem and post mortem inspection and found fit for human consumption. Bovine Spongiform Encephalopathy (BSE) Central Nervous System Cattle oral ID 50 (Co ID 50) Cattle orald LD 50 (Co LD 50) Infectious 50% (ID 50 ) Dose Meat and Bone Meal (MBM) Pithing A transmissible spongiform encephalopathy (see below) of adult cattle. Contamination of MBM in feed with prions is considered to have caused the BSE epidemic that originated in the late 1980s in the UK. The portion of the vertebrate nervous system consisting of the brain and spinal cord (Biology on line: The oral dose which infects 50% of cattle in an experimental test. The oral dose which kills 50% of cattle in an experimental test. The dose which infects 50% of animals in an experimental test. In Commission Directive 92/87/EEC of 26 October 1992 is defined as: Product obtained by heating, drying and grinding whole or parts of warm-blooded land animals from which the fat may have been partially extracted or physically removed. The product must be substantially free of hooves, horn, bristle, hair and feathers, as well as digestive tract content. (1) Products containing more than 13 % fat in the dry matter must be named as 'rich in fat'. It is used as a protein source in animal feed. Slaughtering technique, defined in Commission Decision 2000/418/EC as: laceration of central nervous tissue by means of an elongated rodshaped instrument introduced into cranial cavity. The EFSA Journal (2007) 596, 43-45

44 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association Term Prion Processed Animal Protein (PAP) PrP sc PrP TSE TSE Rapid Tests Scrapie Definition Neologism for proteinaceous infectious particle, frequently used as designation for the infectious agent of TSEs (see below). All known prions contain misfolded isomers of a normal cellular protein (PrP c ). Aggregates of the misfolded protein of sufficient quantity and size are usually associated with TSE infectivity and neurodegenerative diseases in both animals and humans. According to the methodology used for detection of the disease associated, misfolded protein, different terms have been used for its destination (see below). Currently the preponderant hypothesis concerning prions considers that the misfolded protein is the only component of the infectious agent of TSEs. However, a part of TSE experts believe that the protein-only theory has not been proven beyond question. In Commission Regulation (EC) No 829/2007 of 28 June 2007 is defined as: animal protein derived entirely from Category 3 material, which have been treated in accordance with Chapter II of Annex VII so as to render them suitable for direct use as feed material or for any other use in feedingstuffs, including petfood, or for use in organic fertilisers or soil improvers; however, it does not include blood products, milk, milk-based products, colostrum, gelatine, hydrolysed proteins and dicalcium phosphate, eggs and egg-products, tricalcium phosphate and collagen. It comprises MBM and MM (see above). It is used as a protein source in animal feed. Term originally derived from scrapie associated PrP, but also more generally used in all TSEs. Abnormally folded prion protein that has a gradient of resistance to proteinase K digestion. It is associated with infectious potential and with prion disease even in circumstances where it may be sensitive to proteinase K digestion. TSE associated, abnormally folded protein. It is used synonymously with PrP sc or PrP d. Sometimes TSE is replaced by the acronym of the respective disease, e.g. PrP CJD, PrP GSS, PrP BSE, PrP sc, PrP CWD, etc The analysis methods defined by letter (l), point 1., Article 3 of the Regulation CE 999/2001 and for which the results are known within 24 hours. Designates a natural transmissible spongiform encephalopathy (see below) of sheep and goats. This term covers a large variety of agents (TSE strains) with different biological properties. Scrapie has been described in many parts of the world. Can be transmitted naturally or experimentally to other animals such as mice. Is the main prion source for experimental models of TSEs. The EFSA Journal (2007) 596, 44-45

45 Opinion on a TSE risk assessment of the use of bovine spray dried red cells in feeds for fish, in consideration of a report produced by the European Animal Protein Association Term Specified Risk Material (SRM) Transmissible Spongiform Encephalopathy (TSE) Definition The following tissues of animals whose origin is in a Member State or third country or of one of their region with a controlled or undetermined BSE risk according to the Reg. CE 999/2001 As regards bovine animals: (i) the skull excluding the mandible and including the brain and eyes, and the spinal cord of animals aged over 12 months; (ii) the vertebral column excluding the vertebrae of the tail, the spinous and transverse processes of the cervical, thoracic and lumbar vertebrae and the median sacral crest and wings of the sacrum, but including the dorsal root ganglia of animals aged over 24 months; and (iii) the tonsils, the intestines from the duodenum to the rectum and the mesentery of animals of all ages. As regards ovine and caprine animals: (i) the skull including the brain and eyes, the tonsils and the spinal cord of animals aged over 12 months or which have a permanent incisor erupted through the gum, and (ii) the spleen and ileum of animals of all ages. A family of slowly progressive and ultimately fatal diseases of the central nervous system. They are characterized by transmissibility with a long incubation period, and spongiform degeneration of the central nervous system without inflammation and immunity response. Examples in humans include CJD and kuru. Among animals: scrapie and BSE. A synonym for TSE is prion disease. The EFSA Journal (2007) 596, 45-45

46 Final 3 April 2007 THE CASE FOR REINTRODUCING SPRAY-DRIED RED CELLS (SDRC) FROM CATTLE PASSED FIT FOR HUMAN CONSUMPTION INTO THE DIET OF FARMED FISH IN THE EUROPEAN UNION Including a TSE risk assessment comprising the source, process and use of SDRC Prepared in association with: Ray Bradley CBE Private BSE Consultant, Guildford, UK raybradley@btinternet.com And Javier Polo Animal Blood Products expert javier.polo@ampc-europe.com The European Animal Protein Association MARCH 2007

47 EXECUTIVE SUMMARY This document is presented by the European Animal Protein Association (EAPA). The EAPA represents companies that specialise in the production and supply of high quality proteins derived from the blood of animals slaughtered and passed fit for human consumption in the European Union (EU). These blood products satisfy the definition provided in Annex 1, point 4 of the Regulation EC 1774/2002. In practice some of these blood products from nonruminant animals and produced by EAPA members are currently used in human food, feed for food producing animals, including farmed fish for human consumption, pet animals and in pharmaceuticals for human use. This document is concerned only with spray-dried red cells (SDRC) derived from cattle passed as fit for human consumption and their use in farmed fish diets. As a result of the occurrence of BSE in European cattle from 1988 and the occurrence of vcjd in humans from 1996, a Council Decision (2000/766/EC) introduced a temporary ban, from 1 January 2001 until 30 June 2001, on the feeding of processed animal protein (PAP) to farmed animals kept for food including fish. In the period until March 2007 the ban has been partially lifted subject to defined conditions of production and use for certain products but not for bovine SDRC, despite it being conditionally approved for use in human food, pharmaceutical products and as a fertiliser. Apart from the above feed ban, the other main measures to protect public and animal health from the risk of exposure to the BSE agent are a specified risk material (SRM) ban (that protects all species), Rapid testing all slaughter cattle over 30 months old (and risk animals over 24 months old) for BSE and various measures to effectively and safely control the processing, use, destruction and disposal of animal material. The EAPA supports the introduction of these measures and indeed the completeness of the 2001 temporary feed ban but now believes that circumstances have improved so much that bovine SDRC can be reintroduced into farmed fish diets without fear of slowing or endangering the objective of elimination of BSE in the EU. In fact, in the EU there is a continuous year on year reduction in the occurrence of detected BSE infections and cases. The annual total number of positive BSE animals is now low, especially in slaughter animals, thus testifying to the effectiveness of the introduced measures It is further noted that naturally occurring transmissible spongiform encephalopathies (TSE) do not occur in non-ruminant food animal species including fish. Furthermore, one of the constraints to returning to feed rules of earlier times has been the absence in 2001 of effective methods to detect cross-contamination of animal feed with prohibited ruminant protein. Effective, validated and officially accepted methods have now been developed and are used. As a result the use of certain food animal products (including blood products from non-ruminants) has been authorised for use in the diets of nonruminant species and farmed fish in the EU. As a result of the feed bans there has been an adverse environmental impact because surplus bovine blood previously used partly for feed must now be 2

48 destroyed or disposed of in a safe manner either by incineration or processing followed by burial in landfill according to the law. This is an added expense, is harmful to the environment, adds to CO 2 emissions which contributes to global warming and is without added benefit other than improved safety. This document lays out the sequence of legislative events concerning animal feed control in the EU in the BSE era and especially since 2000 until 2007, demonstrates the safety of bovine SDRC by means of a risk assessment covering the hazard (agents causing BSE and atypical BSE), source of bovine blood, collection and processing of the blood to make SDRC, demonstrates the environmental impact of the current ban and presents a case for the return of bovine SDRC to the diet of farmed fish without jeopardising BSE elimination in the EU or endangering the health of consumers. The recipients of this document are requested to urgently review the science relating to any TSE risks in bovine blood from EU cattle passed fit for human consumption and the information presented here by the EAPA with a view to amending EC legislation to permit SDRC into the diet of farmed fish as soon as is practically possible. Furthermore, if this proposal is adopted then the environmental impact of disposal would be lessened, the costs to the farmer and consumer would be reduced and the EU livestock industry would have the current distortion of competition in the global livestock sector removed, thus regaining its earlier competitive edge. The proposals are in line with the general proposals of the European Commission (EC) as expressed in the TSE Roadmap in

49 CONTENTS Executive summary 2 Contents 4 Introduction 7 Sequence of legislative events regarding animal feed in the EU 9 Background 1986 onwards 1988 onwards 1996 onwards Prohibition of SDRC in fish feed onwards 2002 onwards onwards 2005 onwards onwards Other factors 15 Fish welfare Species barrier 16 Environmental factors Conclusions on legislation and related factors Background information for a risk assessment 18 GBR assessment Species TSE risk 19 The TSE agent strain risk Infectivity in blood from sheep 20 Infectivity in blood from cattle 21 4

50 Inherent risks in circulating blood Cross-contamination general 22 Cross contamination stunning and pithing Cross-contamination other sources 23 WHO OIE 24 Conclusion on infectivity in bovine blood TSE risks in additions to blood Blood collection and other abattoir procedures Processing to prepare the red cell fraction 25 Effect of filtration and centrifugation Filter washing and waste disposal 26 Brief risk analysis for SDRC 27 The hazard The starting material and end product Source Species and risk Geographical source and risk TSE agent strain risk TSE risk in the tissue (bovine blood) 28 TSE risk resulting from cross-contamination The TSE risk from additions to the blood 29 Processing bovine blood to make SDRC The use of SDRC Conclusion 30 5

51 Environmental issues resulting from disposal of unwanted or unsafe animal products 31 General Bovine blood Organic potential contamination of blood as biological material Maximizing the value of blood and adding value 32 Final conclusion Request References 33 Annexes 37 Annex 1 - European Animal Protein Association 38 Annex 2 - Assurance procedures to guarantee the safety of spray-dried bovine red cells 39 Annex 3 - Quality procedures: HACCP, ISO Annex 4 - A description of the process to prepare bovine SDRC (as prepared by the EAPA) 43 6

52 INTRODUCTION This document is presented by the European Animal Protein Association (EAPA). Founded in 1988, the EAPA represents companies that specialise in the production and supply of high quality proteins derived from the blood of animals slaughtered and passed fit for human consumption in the European Union (EU). These blood products are embraced by the definition provided in Annex 1, point 4 of the Regulation EC 1774/2002 (EC, 2002) namely: Blood products means products derived from blood or fractions of blood in accordance with Annex VII, Chapter III (specific requirements for blood products) and intended for animal consumption or organic fertilisers. The blood products referred to in this presentation satisfy this definition. In practice high quality, safe blood products produced by EAPA members are used in human food, feed for food producing and pet animals, farmed fish for human consumption and in pharmaceuticals for human use. In this document we are concerned only with the source material namely, Category III bovine blood as specified in Article 6, points a and b of Regulation 1774/2002 (EC, 2002) i.e.: blood from animals that have been passed fit for human consumption in accordance with Community legislation but which is not intended for human consumption for commercial reasons; blood from animals which is rejected as unfit for human consumption but which is not affected by any signs of diseases communicable to humans or animals and derives from carcases that are fit for human consumption in accordance with Community legislation. and spray dried red cells (SDRC) prepared from this blood, as defined later in this document, to support the case for the re-introduction of currently banned bovine SDRC into the diets of farmed fish for human consumption. Bovine SDRC has been used in farmed fish diets for many years including during the height of the BSE epidemic without risk of any kind. However, with the advent of bovine spongiform encephalopathy (BSE) from 1986 onwards throughout the EU and more particularly the occurrence of variant Creutzfeldt- Jakob disease (vcjd) in humans from 1996 onwards in several European and other countries affected with BSE, steps have been taken to eliminate BSE from the European cattle population. This is because vcjd is a distressing and fatal human disease mostly of young people and is primarily caused by the consumption of BSE infected cattle products, especially specified risk material (SRM) or other carcase parts contaminated with SRM. SRM comprises those tissues of ruminant animals that, in a TSE-affected animal, harbour the infectious agent (SSC, 1997). The main procedures introduced to protect public and animal health from the risk of exposure to the BSE agent are an SRM ban (that protects all species), a temporary ban on feeding processed animal protein (PAP) to farmed food animals (including fish), testing all slaughter animals over 30 months old (and risk animals over 24 months old) for BSE and various measures to effectively and safely control the processing, use, destruction and disposal of animal material. The EAPA supports the introduction of these measures. 7

53 As a result of the feed bans there has been an environmental impact because products previously used for feed must be destroyed or disposed of in a safe manner, either by incineration or processing followed by burial in landfill according to the law. This is an added expense without added benefit other than improved safety. The EAPA notes that the feed ban was intended to be temporary. The BSE situation in the EU is showing a continuous year on year reduction in the occurrence of detected BSE-infections and cases and the annual total number of positive BSE animals is low, thus testifying to the effectiveness of the introduced measures. It is further noted that naturally occurring transmissible spongiform encephalopathies (TSE) do not occur in non-ruminant food animal species including fish. Furthermore, one of the constraints to returning to feed rules of earlier times for the latter species has been the absence of effective methods to detect contamination of animal feed, particularly with prohibited ruminant protein. Suitable methods have now been developed, validated and used. As a result the use of certain food animal products (including blood products from non-ruminants) has been authorised for use in non-ruminant species and fish in the EU. The EAPA now presents a scientific case for the removal of the current restrictions from feeding SDRC prepared from cattle passed fit for human consumption, to farmed fish, without jeopardising the objective of BSE elimination from the EU or endangering the health of the consumer. Furthermore, if this proposal is adopted then the environmental impact of disposal would be lessened. Also the costs to the farmer and consumer would be reduced and the EU livestock industry would have the current distortion of competition in the global livestock sector removed, thus regaining its earlier competitive edge. This is in line with the general proposals of the European Commission (EC) as expressed in the TSE Roadmap (EC, 2005a). The purpose of this document is to: lay out the sequence of legislative events concerning animal feed control in the EU in the BSE era and especially since 2000 demonstrate the safety of bovine SDRC by means of a risk assessment demonstrate the environmental impact of the current ban and to present a case for the return of bovine SDRC to the diet of farmed fish without jeopardising BSE elimination in the EU or endangering the health of consumers. 8

54 SEQUENCE OF LEGISLATIVE EVENTS REGARDING ANIMAL FEED IN THE EU Background 1986 onwards BSE in domestic cattle was first recorded in the UK in November 1986 (Wells et al, 1987). By the end of 1987 it had been established beyond reasonable doubt that BSE was a disease transmitted by feed carrying the BSE agent, the vehicle being meat-and-bone-meal (MBM) one of the two main products of rendering (Wilesmith et al, 1988). Rendering is a commonly used industrial cooking process used to convert ground animal carcase waste material into useable products namely greaves (mainly protein and mineral material) and tallow (fat). Greaves is ground to produce MBM. Blood especially from pigs and cattle has a significantly higher value than MBM and that not used directly in human food is converted into high quality blood products including SDRC and spray-dried plasma by specialist companies such as those belonging to the EAPA or, into blood meal mainly using a heat process onwards The precise origin of BSE is not known and may never be known with certainty. However, once infection was introduced, the UK epidemic was fuelled by the recycling of BSE-infected tissues (now called SRM) from infected and diseased cattle via MBM. SRM from certain ages of cattle comprises the skull and vertebral column, central nervous tissue (brain, spinal cord, associated ganglia and the eye in which the majority of infectivity resides) and certain other lymphoreticular tissues including the intestine from duodenum to rectum inclusive. The UK introduced a ban on feeding ruminant protein to ruminant animals in 1988 and a ban on the use of SRM (then called specified bovine offal - SBO) in human food in 1989 and in the feed for all species in The EC responded with an EU-wide ruminant feed ban in July onwards In March 1996 the Spongiform Encephalopathy Advisory Committee of the UK (SEAC), on receiving information about ten cases of a new variant form of CJD (vcjd) amongst other things, recommended the government to extend the ruminant feed ban to protect all species of farmed food animal including horses and fish by prohibiting the use of mammalian protein in their feed. The reason was that the original feed ban, though sound in concept and very good, was imperfect, resulting in leakage of MBM legally present in fish and poultry rations into ruminant rations. The SEAC advice was implemented on 29 March Some other countries introduced similar but generally less stringent national legislation. European Commission Decision 2000/418 introduced the EU-wide SRM ban in June During the whole period of the BSE epidemic until 31 9

55 December 2000, bovine SDRC could be fed legally to farmed fish throughout the Union. From then things changed. Prohibition of animal proteins in feed for farm animals including fish 2001 onwards From 1 January 2001 Council Decision 2000/766/EC (EC, 2000) was operative, resulting in a temporary ban until 30 June 2001 on the feeding of processed animal protein (PAP) to farmed animals kept for food including fish. In this instance PAP was defined to encompass most animal-derived proteins including any derived from blood like SDRC. There were defined exceptions for fishmeal (for feeding to non-ruminants only), gelatine, di-calcium phosphate and milk products. Furthermore all PAP intended for feeding to farmed animals had to be withdrawn from the market, collected and destroyed according to existing legislation. The purpose of this legislation was to reduce, or eliminate, the risk that ruminant animal feed was cross-contaminated with PAP and potentially BSE agents. Commission Decision 2001/9/EC (EC, 2001a) specified how fish meal (amongst other things) could be marketed and fed to non-ruminant animals including fish. The EC measures regarding animal feed control were consolidated in Regulation (EC) No 999/2001 (EC, 2001b) that laid down the rules for the prevention and elimination of certain TSE. In Article 7 (1) of the Regulation it states that feeding to ruminants of proteins derived from mammals is prohibited. Article 7 (2) specified that the prohibition is more extensive still in geographical BSE risk (GBR) CATEGORY 5 countries, none of which now exist in the EU. There are some exceptions to the prohibitions. For example, dried plasma and other blood products (with the exception of bovine blood products for feeding to ruminants) are permitted for use as specified in Annex IV, point 2e to the Regulation, Thus the road would now be open at some point in the future to permit the feeding of fish protein to ruminants and of bovine blood products to non-ruminants including to fish, but requires a change to the legislation. Importantly in paragraph 11, in regard to feed controls the Regulation specifies they should be proportionate to the risks involved. The Regulation (EC) No 999/2001 (EC 2001b) entered into force on 1 June 2001 and applied from 1 July However, the latter was not possible because before that could happen EU Member States had first to be classified on the basis of a GBR assessment that had not then taken place by the due date. Therefore transitional arrangements were necessary to deal with this temporary problem. The transitional arrangements were specified in Commission Regulation (EC) 1326/2001 of 29 th June 2001 (EC, 2001c). These determined (in Articles 1 and 2) that the 1 July date was postponed at least until I January 2002 and thereafter until the entry into force of the Decision determining the BSE status of that Member State and until the Community provisions on animal feeding relevant to transmissible spongiform encephalopathies are effectively enforced there. 10

56 Commission Regulation (EC) 1326/2001 Annex III A point 4 also bans the pithing of ruminant animals throughout the EU (EC, 2001c). Included in Regulation (EC) No 999/2001 was the requirement for monitoring cattle for evidence of BSE using approved Rapid tests performed in approved national laboratories. All slaughter cattle over 30 months of age are routinely tested and any positives and close neighbours on the slaughter line (e.g., the one before and two after on the line) are treated as SRM and destroyed. Risk animals for slaughter over 24 months of age are similarly tested and failures similarly destroyed. In 2005 (the latest year for which data are available) 113 positive BSE cases were identified in EU healthy slaughter cattle following the testing of over 8.5 millions (EC, 2006). No positive animals entered the food or feed chain onwards An animal by-products Regulation (EC No 1774/2002) was introduced in 2002 by the European Parliament and the Council of Agriculture (EC, 2002) and was applicable from 1 May This Regulation separated animal by-products into one of three Categories. Category 3 animal by-products (including blood products) can only be derived from healthy animals, including cattle that have been officially inspected ante- and post mortem and have been passed fit for human consumption. Furthermore, in Annex VII of the Regulation (including CHAPTER 3 thereof) there are specified hygiene requirements for the processing and placing on the market of PAP and blood products that could be used as food material. All the blood collected and processed into blood products by EAPA members, including bovine SDRC, satisfy the conditions of the Regulation. PAP is defined in Annex 1, Paragraph 43 but this definition does not include blood products. These are defined in Paragraph 4: Products derived from blood or fractions of blood, excluding blood meal: they include: dried/frozen/liquid plasma, dried whole blood, dried/frozen/liquid red cells or fractions thereof or mixtures. Consequently, the restriction on use for intra-species feeding animal proteins specified in Article 22 of the Regulation, whilst applying to PAP, does not apply to blood products. Nevertheless this EAPA document seeks currently only to feed bovine SDRC to fish and in so doing there would be no difference from the compliance for PAP as specified in Paragraph 1a of Article onwards It is important that legislation is enforced and that the mechanisms by means of which this is done are reliable, practical, acceptable and effective. It is important that tests to detect the species of origin of PAP are available, validated and effective in use. Analytical methods to determine the constituents of animal origin (including fish material and blood are specified in Commission Directive 2003/126/EC (EC, 2003b). It is now possible though difficult, to differentiate between fishmeal and other PAP posing a TSE risk and accordingly it was proposed in Commission Regulation (EC) No 1234/2003 (EC, 2003a) that the earlier Commission Decision 2001/9/EC should be simplified. Evidence to 11

57 support the introduction of Regulation came from the Opinion of the Scientific Steering Committee (SSC) of 17 September 1999 that there is no evidence of the natural occurrence of TSE in non-ruminant farmed animals producing food such as pigs and poultry (SSC, 1999) or fish (Ingrosso et al, 2006; EFSA, 2007) onwards Commission Regulation (EC) No 1292/2005 of 5 August 2005 (EC, 2005b) recommended that the restrictions on feeding to farm animals of blood products and hydrolysed proteins from non-ruminant species should be relaxed because farmed food animals of these species have shown no natural occurrence of TSE. Furthermore, in paragraphs (3) it states that the performance of laboratories for detecting small amounts of mammalian proteins in feedingstuffs using methods itemised in Commission Directive 2003/126/EC (EC, 2003b) has improved considerably. In paragraph (6) of 1292/2005 it further states that given that controls on the ban on animal proteins are based on the detection of bones and muscle fibres (also blood, (EC, 2003b)) blood products from nonruminants should not jeopardise controls on the presence of potentially TSEinfected proteins. Therefore, the restrictions on feeding to farmed animals of blood products and hydrolysed proteins from non-ruminants should be relaxed. Accordingly the Regulation amended Annex IV of Regulation No 999/2001 of the European Parliament and of the Council as regards animal nutrition and in regard to blood products as follows: First it extends the prohibition provided for in Article 7(1) of blood products to all farmed animals (except carnivorous fur producing animals) and to ruminant animals of animal protein or feedingstuff containing animal protein. Second it permits derogations so that the prohibitions provided for in Article 7(1) and (2) shall not apply to the feeding to non-ruminant animals: 1. Of fishmeal in accordance with conditions laid down in Point B, 2. Of blood products from non-ruminants in accordance with the conditions laid down in Point D 3. Of blood meal derived from non-ruminants to fish also in accordance with the conditions laid down in Point D Point B requires complete separation the production, transport and use of fishmeal and feedstuffs containing fishmeal at all times and appropriate labelling to clearly indicate the feed must not be fed to ruminant animals. Point D similarly requires separation of non-ruminant blood and blood products from ruminant materials at all times from slaughter to finished feed and regular sampling and analysis of blood, blood products and blood meal of non-ruminant origin to detect the presence of ruminant proteins. There must also be appropriate labelling to clearly indicate the feed must not be fed to ruminant animals. 12

58 It is noted that in 2007 when the BSE situation has improved still further and the risk from it in feed is significantly diminished, a similar case could be upheld for the conditional reintroduction of bovine SDRC into the diets of farmed fish 2006 onwards In the Opinion of the European Economic and Social Committee (EESC) on the disposal of animal carcasses and the use of animal by-products (EESC, 2006) it is noted as follows: Safety standards for food production are much higher in Europe than in third countries, but they do guarantee food safety for consumers, environmental protection and animal welfare. Maintaining these standards, with the higher production costs that they entail, will only be possible if production continues in Europe In a nutshell, the conclusion that we can draw from all of these scientific studies is that no epidemiological proof exists for the theory that pigs, poultry or fish are susceptible to contracting BSE or that these species have been affected by BSE. To date, no scientific tests have demonstrated the development of TSE in pigs, poultry or fish. 4.3 Economic impact on the use of animal by-products Banning the use of animal protein in feedingstuffs for pigs, poultry and fish has led to significantly higher production costs in Europe and has consequently caused further problems of distortion of competition vis-à-vis other countries such as Brazil, Argentina and the USA, for example, where the use of animal protein is authorised. These higher costs have had consequences at various levels, with slaughterhouse by-products no longer being a benefit, because they now entail destruction costs, and with increased demand for vegetable protein leading to higher prices and consequently higher feed prices (8) The main issue is to ensure that there is no cross contamination in meat meal In any event, regardless of the above comments, there is now a real opportunity to set up a mechanism to monitor systems exclusively supplying pork protein for poultry feed and vice versa, because: meat meal from pigs and meat meal from poultry can never be produced at the same slaughterhouse, because these species require different slaughtering facilities; since some plants produce only poultry feed and others only pig feed, the two can never be accidentally mixed; the same applies to plants that have separate production lines for different species. 13

59 From the above, notwithstanding the presumed desire by the EESC to have MBM reintroduced in pig and poultry feed with a species barrier (which is not the purpose of our document) it can be concluded that: 2007 Food and feed safety, environmental protection and animal welfare (discussed briefly below) are each important issues Cross-contamination of ruminant rations with TSE-risk materials as determined by various EC Regulations must be prevented Non-ruminant animals do not succumb naturally to any form of TSE and are at a low risk of contracting TSE especially across a species barrier and by the relatively inefficient oral route. As stated in Regulation 999/2001 (EC, 2001b) feed prohibitions should be proportionate to the risks involved. As time has passed the TSE risks especially since 2001 have significantly reduced in the whole of the EU and at a certain point (as in 2007 for bovine SDRC as defined herein) are sufficiently low to permit a conditional return to previous use in farmed fish diets. The European Food Safety Authority (EFSA) and its Scientific Panel on Biological Hazards was invited by the European Parliament to provide an opinion (EFSA, 2007) on the state of play as regards the health risks of the feeding of ruminants with fishmeal in relation to the risk of TSE and if this could have negative consequences in terms of public health. The experts of the Scientific Panel on Biological Hazards concluded that any risk of generation and/or amplification of TSE in fish is remote. So the risk (if any) would be from cross contamination of the fishmeal. This could occur in two ways: first by contamination of the fishmeal after production with MBM or diets containing MBM, and second from ruminant protein being present in the alimentary tract contents at death of the fish as a result of the fish feed containing a mammalian protein. The latter would only be a risk if farmed fish waste was itself converted to fishmeal and mixed with fish meal prepared from free-living marine fish. There are methods available to detect contamination of fishmeal and other heat-treated animal protein with MBM by identification of species-specific DNA though proof of absence will never be possible. A number of additional points are made in the EFSA document: On the question whether the feeding of wild fishmeal to farmed fish presents any risk to animal or human health vis-à-vis TSE, there was currently no evidence of any such risk existing. A revision of Regulation (EC) No 999/2001 (EC, 2001b) is foreseen for the end of 2006; the current draft revision allows feeding young ruminants with fishmeal and introduces a tolerance level for fishmeal in feed for adult cattle under strict conditions (EFSA, 2007). In 2006 the number of incidents of animal feed being illegally contaminated with animal proteins or bones in the EU was 6. MBM is likely to be the only source of cross contamination of fishmeal. 14

60 There is no evidence for the replication of TSE agents in fish. The strength of a species barrier between fish and mammals is likely to be large, Microscopy is capable of detecting 0.5% of MBM in animal feed that also contains 5% of fishmeal (sensitivity about 95%).Sensitivity drops to about 70% when feed contains 0.1% of MBM. Tests to detect species specific DNA using the polymerase chain reaction in feed containing heat-treated MBM and fishmeal are more sensitive than using microscopy alone. There is a potential hazard of residual TSE infectivity in fishmeal produced from fish experimentally fed (up to 15 days before death) with TSE contaminated feed. Authorised ingredients of the same species to the species being fed like milk protein and tallow (and presumably blood products) can contribute DNA from the source species and so make a test positive even though the feed is constituted according to the law. Intensively fed farmed fish in the EU are carnivorous and have a high requirement for animal protein and fat such as that provided by fishmeal and fish oil in their diets. The fishmeal is predominantly produced from a variety of ocean-caught marine fish. Salmonids have a requirement for omega-3 (n-3) fatty acids of longer chain lengths and certain amino acids. Consequently, the most important ingredient in the diets of farmed fish is fishmeal. Fishmeal is derived from whole dead wild caught fish or trimmings of such fish after filleting for human consumption (European Parliament, 2004). Trimmings typically could make up about 33% of the starting material but in some countries it could be up to 100% or as low as 10%.The method of production is a continuous wet reduction process, which does not denature proteins and would have little, if any, effect on TSE agents. However, other conventional organisms like bacteria and viruses would be inactivated or destroyed. The typical inclusion rates for fish meal in animal diets are around 2-10% for terrestrial animal species, but can to rise to in excess of 40% for fish diets (European Parliament 2004), Other factors Fish welfare SDRC made an important contribution to the diet of farmed fish until the ban on use of animal proteins in the farmed food animals was put in place on 1 January The species of fish selected for fish farming because of their high nutritional value in the human diet, high palatability, inherent safety and fast growth rate, are carnivorous. Wild fish of these species would consume other fish and crustaceans as part of their natural diet. Thus it is natural for farmed fish to be fed with animal protein and prior to the EU-wide ban on feeding animal protein to farmed food animal species it was entirely natural to use both 15

61 fishmeal and bovine SDRC in their diet. The latter has a high (> 90%) digestible protein content. At that time it was also favoured by the industry because of the absence of any risk from dioxins that might otherwise have been present. It is just as logical now to return to the former practice by permitting the use of SDRC back into farmed fish diets particularly as it introduces an additional safety factor in regard to TSE, namely a natural and probably large species barrier. Species barrier The species barrier is a natural barrier to the transmission of TSE agents from one species to another. Use is made of this in the Regulations pertaining to TSE because the feeding of animal protein from one food animal species is not permitted to be fed to the same species with certain well defined exceptions such as for milk and milk products (EC, 2002). In regard to fish, fishmeal is generally produced from wild marine fish of mixed species and it is regarded as safe to feed such material to farmed fish. Thus any risk in marine fishmeal fed to other food animals than fish is remote in the extreme because it is expected that there would be a large species barrier between fish and mammals including those like ruminants that can be affected with TSE. It is already possible under current Regulations to feed fishmeal to non-ruminant species. It is also possible to feed blood products from nonruminant species such as pigs to non-ruminant species. In the case of blood products from pigs spray-dried plasma from Category 3 pigs is permitted even in the rations for pigs because there are extenuating circumstances for this need (e.g., provision of immunoglobulins) and the need is only for young pigs and not for breeding or fattening pigs. This is permitted also because a TSE risk assessment has shown any risk in pig blood (which is also used for human consumption) is negligible, even for pigs, where there is no species barrier. Environmental factors Although bovine blood has a variety of uses there is a large surplus if it is not used in feed. Under the Regulations this surplus has to be completely destroyed or made safe for disposal with environmental consequences. These aspects will be discussed in a subsequent section of this document. CONCLUSIONS ON LEGISLATION AND RELATED FACTORS Currently EC Regulations prohibit the use of most PAP including blood products from ruminant animals being used in feed of any species. From 2001 the restrictions applied to almost all PAP from any species being used in any species of farmed food animal even though some were regarded as having a negligible TSE risk. The latter were not permitted partly because methods to detect ruminant protein (where the TSE risk would be highest) in heat treated PAP were insufficiently developed to distinguish them from other (safe) proteins. Since then the BSE situation has improved and continues to do so, 16

62 new tests have been developed to identify the species of origin of the PAP and the political and consumer climate is such that some of the original controls have been relaxed, particularly in regard to feeding non-ruminant species including fish with certain previously prohibited products including blood products from non-ruminants. Marine fishmeal has been authorised for the feeding of non-ruminants and farmed fish and there are proposals to permit it into at least some rations of young bovine animals. The current Regulations make provision for a progressive relaxation of the current controls that restrict the feeding of certain protein products to certain species as part of an on-going process in line with the guidance in the EC TSE Roadmap (EC, 2005a) and subject to scrutiny by the appropriate scientific committee. The scene is set for permitting the conditional use of bovine SDRC in the diet of farmed fish. The case is supported by the fact that fish do not suffer from nor replicate TSE agents and fishmeal from farmed fish is not used in the diets of cattle thus breaking the chain of any infection if it was present. The next section will demonstrate clearly that there is a negligible risk of TSE infectivity ever being present in bovine SDRC derived from EU slaughter cattle. Finally it will be shown that the current adverse effects on the environment from disposing of surplus cattle blood would be significantly reduced if bovine SDRC were again permitted into farmed fish diets as has been the case in the past with considerable beneficial effect and no occurrence of TSE. 17

63 BACKGROUND INFORMATION FOR A RISK ASSESSMENT Bovine SDRC are solely sourced from cattle in the EU and, so far as this document is concerned will be fed only to farmed fish. Thus the assessment of the TSE risk in the product (SDRC) will depend on the following factors: The Geographic BSE risk (GBR) in the country of origin The TSE risk in cattle (species risk) The TSE agent strain risk The TSE risk in the tissue (bovine blood) The TSE risk resulting from cross-contamination The TSE risk from additions to the blood The collection procedure The processing procedure and The use (feeding to farmed fish). Each of these factors will be discussed in turn. GBR ASSESSMENT The EFSA has Published (EFSA, 2006) a list of countries for which a GBR assessment has been completed, either by the SSC, or by the EFSA in the period Since BSE is a dynamic disease, the GBR is also dynamic and requires continual updating to ensure it is accurate at the time in question. Some countries have been assessed more than once and some not at all. Some have not been assessed since 2000 and the BSE situation has significantly changed since then. EAPA members process bovine blood mainly from cattle in their native country but under EU law cattle are conditionally permitted to move from one country to another with appropriate identification and documentation. Most EU Member States that have been GBR assessed fall into Category III (BSE likely but not confirmed, or confirmed at a lower level). EAPA members are in Belgium, Denmark, France, Germany, Italy, Poland, Spain, Sweden, the Netherlands and the United Kingdom. All these countries have reported confirmed cases of BSE. All these countries have been classified as Category III except for Sweden that was last classified in 2004 as Category II (BSE unlikely but not excluded) Sweden has since reported a case of BSE so should therefore, now be in Category III. The UK was last classified in 2000 as Category IV (BSE confirmed at a higher level). However, since then, the BSE situation has improved greatly and earlier restrictions on trading have now been removed and the BSE risk is now regarded as equivalent to that in other EU Member States. For practical purposes the GBR in all EU Member States is now very similar and could reasonably be regarded as not worse than Category III. This is assumed in this document. The Office International des Epizooties (OIE) is also known as the World Organisation for Animal Health and produces an annual publication known as 18

64 the International Animal Health Code for terrestrial animals in which there is a chapter ( ) on BSE (OIE, 2006). The intention of the recommendations in the chapter is to enable effective management of TSE risks in cattle and cattle products especially in regard to international trade. Following a risk assessment conducted in the manner indicated, countries can be classified into one of three categories: Negligible BSE risk Controlled BSE risk and Undetermined BSE risk It is anticipated that all EU Member States (including all countries where the EAPA members collect bovine blood) will be classified as BSE controlled risk countries. By July 2007 it is anticipated that the EFSA and OIE categories will be aligned in a manner to be determined. Species TSE risk Naturally occurring TSE occurs in primates, ruminants (including cattle), Felidae and Mustelidae. The only animal tissues used in the preparation of SDRC are from domestic cattle (Bos taurus). It is highly unlikely, but cannot be excluded completely, that an occasional Bos indicus or Bos indicus cross could be included in the source animals. However, on current knowledge, this would not result in any higher risk of BSE in blood if the animal was infected, provided all the other conditions were satisfied. The risk of transmission of TSE is generally greatest within species (but it can be modified by genetic variation especially in the PrP gene) whereas between species transmission is usually reduced due to the presence of a transmission barrier. This is also referred to as the species barrier. The range of sequence homology for PrP between fish and mammalian species is lower than 40%, suggesting a high species barrier for transmission of mammalian prions to fish (EFSA, 2007). It is reasonable to assume therefore that there is a significant species barrier between cattle and fish that will naturally restrict transmission of TSE from the one species to the other and for infectivity generated in a species, in either direction. The TSE agent strain risk Like bacteria and viruses, TSE agents vary in their molecular and biological properties and so exist as a number of strains. For example, some 20 scrapie agents (responsible for the TSE in sheep and goats called scrapie) have been identified in laboratory experiments. BSE agent on the other hand exists as a single major strain of agent and is responsible for TSE in cattle, vcjd in man, FSE in domestic cats and TSE in captive wild ruminants. BSE is the only naturally occurring TSE of cattle and occurs in at least two forms: classical BSE and atypical BSE, which has only recently been discovered and is rare (c 35 cases worldwide compared with >185,000 cases of classical BSE) and mostly is confirmed in cattle over the age of 10 years. BSE has a peak age of occurrence of about 5 years. Both types have been reported from Europe, Asia and North America. 19

65 There are two molecular forms of atypical BSE called H (high) and L (low) distinguished on Western blots for PrP (Buschmann et al, 2006). Limited transmission studies have shown that the PrP phenotype (H or L) is maintained on sub-passage. There are different pathological phenotypes of atypical BSE. One is called bovine amyloidotic spongiform encephalopathy or BASE (Casalone et al, 2004) where the lesions predominate in the rostral cerebrum as distinct from the brainstem in classical BSE. It is too early to say yet if the biological strain of agents from atypical and classical cases differ, and if so how. We do know that the form of PrP Sc in the diseases is different. Knowledge about atypical BSE including the epidemiology of the disease, the nature of the causal agent, its distribution in tissues and its pathogenicity for humans is still evolving. One possibility is that this disease is the manifestation of a historical, ubiquitous, sporadic form of cattle TSE (Brown et al, 2006) that has only been detected as a result of improved surveillance during the BSE era. If this is the case, to date no evidence has been found to support a risk to human health from atypical BSE. Alternatively atypical BSE may be a new disease like conventional BSE with currently unknown consequences for humans and animals. It is too early yet to say if the measures to control classical BSE will be effective for atypical BSE. However, evidence suggests that atypical BSE can be detected using currently approved tests for the detection of classical BSE in which case there would be no increase in the TSE risk in blood collected to make SDRC because any animal over 30 months old having a positive test for BSE would be eliminated. Atypical BSE has only been reported in one animal less than 30 months old in Japan. Infectivity in blood from sheep Although for many years there has been an uncertainty about blood being infected in humans and dubious evidence that blood had ever been implicated in natural disease transmission in humans, things have changed in more recent times. Evidence has grown from experimental studies that it may be infected in certain species and with certain agents. For example, Houston et al (2000) and Hunter et al, (2002) reported the transmission of natural scrapie and experimental BSE in sheep by transfusion. Although transfusion studies have not been reported in cattle using blood from naturally or experimentally BSEinfected cattle extensive studies in mice and cattle using other routes have shown no detectable infectivity. There has also never been reported any epidemiological evidence of TSE infectivity in bovine blood. In October 2000 the SSC reviewed the recent research and gave an opinion on its implications (SSC, 2000). However, they considered that did not change their earlier opinions of the safety of blood including ruminant blood. It is stressed that not only are sheep slaughtered on a separate line or in a different abattoir from cattle there is no co-mingling of blood from sheep with blood from cattle. Furthermore, EAPA member so not collect sheep blood for any purpose. 20

66 Infectivity in blood from cattle In regard to TSE infectivity in bovine blood there are two aspects to consider. The first is the inherent infectivity present in the tissue as it circulates within the animal before slaughter. The second is any infectivity that might be introduced as a result of stunning, subsequent carcase dressing procedures, collection processes and during anticoagulant addition. Each of these risks will be addressed in turn. Inherent risks in circulating blood To date no tested component of bovine blood, fetal calf blood or whole blood has shown detectable infectivity following bioassay in susceptible mice by the combined i/c, i/p routes (Fraser and Foster, 1994, Bradley 1999) (TABLE 1). Furthermore the spleen (one function of which is to filter blood) from cattle with BSE is devoid of detectable infectivity and PrP Sc (Buschmann and Groschup, 2005: Wells, 2006). Buffy coat collected at 6, 18, 26 and 32 months postchallenge from experimentally dosed cattle has also been bioassayed in cattle by the i/c route (Wells, 2006).. No brains from these cattle showed evidence of PrP Sc by Western blot. Furthermore, no brain lesions were detected following microscopic examination and no PrP TSE was detected by IHC either, though the results for the buffy coat collected at 26 weeks post challenge are pending (Information kindly provided by Mr SAC Hawkins). TABLE 1 Results of bioassay of blood/ blood components from cattle with natural or experimental BSE Tissue Natural BSE in cattle Bioassay i/c + i/p in mice Bioassay i/c in cattle Experimental BSE in cattle Bioassay Bioassay i/c + i/p in i/c in cattle mice Blood clot NDI Serum NDI Buffy coat NDI - NDI* NDI* Fetal calf blood NDI Experiment not done. NDI No detectable infectivity * In buffy coat collected at 6, 18, 26 and 32 months post-challenge. IHC results for the 26 month group are pending. It has been claimed that the use of newly developed, ultra-sensitive, but so far unevaluated, commercial tests for PrP Sc in bovine blood has been able to distinguish between cattle experimentally infected with BSE and those without. Thus there is a prospect that in the future it may be possible to develop an effective live animal test. Several questions arise from this development. The first is, does a positive test correlate with infectivity? The second is at what stage of incubation might the developed test be effective? If a correlation with 21

67 infectivity is determined how much infectivity is present? Blind studies have not yet been conducted and the significance of the early findings is at present unclear. In any event, in the EU, approved Rapid tests for BSE are applied to brain material from all slaughter cattle >30 months old and this procedure is accepted to provide adequate safety (if the test is negative) for humans to consume authorized cattle products including blood. Cross-contamination general The preceding paragraph determines the risk category in an uncontaminated state. It is theoretically possible that any tissue or organ collected from cattle could become contaminated with other tissues especially at the point of collection. To avoid cross-contamination at the time of collection effective procedures must be in place and be consistently enforced to a high standard. No unavoidable cross contamination can be permitted at any time and especially with SRM. Not all bovine tissues in which infectivity has been found are classified as SRM (see for example Buschmann and Groschup 2005 who identified BSE infectivity in the M. semitendinosus and various peripheral nerves following inoculation into highly BSE sensitive transgenic mice). Thus TSE risks, if any, are confined to those risks associated with any inherent infectivity in the tissue itself and with any infectivity that could be introduced at the time of collection. EAPA members take precautions to avoid contact with any SRM at the time of collecting the blood. Cross contamination stunning and pithing For welfare reasons most cattle are stunned prior to killing by bleeding out, using some form of stunner. Certain types of penetrating stunners have been shown to generate brain emboli (Garland, Bauer and Bailey, 1996) or biochemical evidence thereof (Anil et al, 1999).) in a proportion of stunned animals. Pithing, a practice that has been used in some abattoirs where penetrative stunning is applied, has a similar effect. Risks are considered greatest following stunning with a device injecting air or gas into the cranial cavity, or to a pithing process following any kind of penetrative stunning and are prohibited in countries with a GBR higher than I. Thus in the EU such stunners are prohibited for use in cattle and pithing is also prohibited thus reducing any risk there may be from these sources (EC, 2001b). There is also evidence that conventional captive bolt guns that penetrate the skull and percussion guns that do not penetrate the skull cause embolism of venous blood (Coore et al, 2005). Furthermore, penetrative stunning can result in contamination of the gun, the blood, personnel and environment (Daly et al, 2002). To minimize risks of cross-contamination therefore, it is preferable that blood is collected in an appropriate way from cattle stunned using electrocution, or following Kosher/Halal slaughter methods. Although no reports of investigations to determine if brain emboli or brain protein occur in the bloodstream of cattle following stunning by electrocution or following Halal or Kosher slaughter, it is presumed to be highly unlikely in the absence of significant trauma to the cranium. Thus, it is likely that any risk from these three 22

68 processes would be the lowest achievable in a commercial abattoir situation and negligible. It is accepted that for practical reasons in the EU it may not be possible to use any of these last three methods on a sufficient scale to deliver the quantities of blood required for the production of SDRC. Thus the risk from the use of captive bolt pistols must be included in the assessment. However, the risk in the EU is neutralised because BSE in slaughter cattle is very rare now and getting rarer especially in cattle younger than 30 months old (EC, 2006) and all slaughter cattle >30 months old are tested by the rapid test and failures are destroyed. Cross-contamination other sources Apart from the risk from embolism, if a penetrating stun gun is used then the hole in the skull can permit brain material to ooze from the hole and potentially contaminate blood collected from the severed neck vessels at bleeding out unless steps are taken to prevent it, such as by using a potato starch bung. Blood collections made by EAPA members take precautions to reduce risks from cross-contamination of blood by any other bovine tissues or from environmental sources. Risks of TSE contamination from other specified bovine offal such as the spinal cord or intestine, is most unlikely because these tissues are only exposed downstream in the slaughter hall and well away from the blood collection point Thus the likelihood of BSE infectivity entering blood collected for SDRC manufacture in the EU is highly unlikely. It is noted that In the EU no authority is concerned that the level of TSE risk in edible cattle tissues (including blood) derived from animals passed fit for human consumption is greater than negligible. Any risk that there may be would be even less for other species like fish where a substantial species barrier exists WHO The WHO held an Expert Consultation on tissue infectivity distribution in TSE on September 2005 (WHO, 2006) to take account of new information since the previous Consultation in The category for blood has not changed. In regard to blood in general, the WHO places this tissue in Table 1B - Lower infectivity tissues because in vcjd in humans and in scrapie in sheep evidence of infectivity has been found (WHO, 2006). However, it is noted that in bovine blood there is absence of detectable infectivity. In establishing the risk of infectivity in specific tissues by research, in most instances very stringent collection procedures (beyond those practical to adopt in commercial slaughter situations) are adopted. OIE The OIE during the Annual Meeting of the International Committee held in Paris in 2006, the Terrestrial Animal Health Code, Chapter on Bovine Spongiform Encephalopathy (OIE, 2006) was approved to state as follows: 23

69 The recommendations in this Chapter are intended to manage the human and animal health risks associated with the presence of the bovine spongiform encephalopathy (BSE) agent in cattle (Bos taurus and B. indicus) only. 1. When authorising import or transit of the following commodities and any products made from these commodities and containing no other tissues from cattle, Veterinary Administrations should not require any BSE related conditions, regardless of the BSE risk status of the cattle population of the exporting country, zone or compartment: h) blood and blood by-products, from cattle which were not subjected to a stunning process, prior to slaughter, with a device injecting compressed air or gas into the cranial cavity, or to a pithing process. Conclusion on infectivity in bovine blood Although blood is a WHO Lower risk tissue, at the current time there is no evidence to support there being any BSE infectivity in bovine blood at any time during incubation or in the clinical phase of disease. Thus blood collected from a living bovine animal that passes ante and post mortem examination (and if >30 months old and passes a Rapid test can be concluded to present a TSE negligible risk. This is endorsed by the OIE who have no restriction on trading in bovine blood collected in the manner described. TSE risks in additions to blood To enable collected blood to be separated during processing into its main components (plasma and cells) anticoagulant is used to prevent clotting. To achieve this, solutions of sodium citrate and/or sodium phosphate are used. These two chemicals are inorganic and are not sourced or prepared from animal material so there is no TSE hazard or risk. Only freshly-prepared solutions made by dissolving the salts in potable water and any containers used are covered to prevent cross-contamination from the environment. Blood collection and other abattoir procedures After the administrative procedures like animal identification (and weighing if appropriate) animals proceed to slaughter and enter individually into the knocking box where they are restrained. The animal is then stunned using a gun that does not inject air under pressure into the cranial cavity. The animal immediately collapses and is released from the knocking box to fall on the floor into lateral recumbency. A hind limb is shackled with a chain so the stunned animal can be raised from the ground with the head down using a hydraulic, electrical or mechanical lift. The hide is opened and a sticking knife is used to sever the major blood vessels in the neck. The released blood drains into a canal or trough (sometimes through a plastic tube incorporated in a hollow 24

70 Knife) and enters a collecting tank where it is mixed with coagulant. The angle at which the carcase is hung enables the blood to enter the tank without being contaminated with any brain or other material. The blood is filtered (mesh size of between 0.42 mm and 1.20 mm, depending on the slaughterhouse) at the slaughterhouse and collected in a refrigerated stainless steel storage tank and chilled to 4 C. The storage tank co-mingles blood from multiple animals. One tank may hold the blood from adult cattle. From here, once approved for removal, the chilled blood is pumped in a sealed system to dedicated, sealed, isothermal, road tankers for transportation to the processing plant. The amount collected is recorded and audited at each end of the journey. All equipment including knives and others articles that contact the carcase or organs are regularly washed and cleaned during slaughter process though it is unlikely that TSE agents would be inactivated if present. Tanks and all other apparatus that is contacted with blood are thoroughly washed, cleaned and sterilized as determined by the Standard Operating Procedures (SOP) of the abattoir and in conformity with meat hygiene regulations. Processing to prepare the red cell fraction All EAPA plants are dedicated to only processing blood from a single species and operate to international standards such as ISO 9000 using HACCP or equivalent quality rules. After discharging the blood at the manufacturing plant it is filtered again (mesh diameter of 0.5 mm to 1.2 mm depending of the manufacturing plant) to remove blood clots.. Before centrifugation the blood is filtered again (mesh size of µm depending of the manufacturing plant). Immediately following centrifugation the plasma and red cell fractions are transferred to separate refrigerated, stainless steel tanks and held at 4ºC. Centrifugation is followed by a final filtration of the cellular fraction (0.14 to 1.00 mm, depending of the type of spray-dry. Filters are cleaned several times a day with clean water. The solid residue goes to waste with the water for treatment and then disposal (see below). Effect of filtration and centrifugation Filtration removes clots and debris greater than the mesh sizes of the filters. If any tissue-associated infectivity was present then it would be removed, if within or attached to the particles collected by the filter. There would be no inactivation of infectivity and any infectivity within the plasma or in the cellular fraction of blood would remain unaltered. 25

71 Centrifugation separates the cells from plasma. The cellular content will be composed of a mixture of red cells (the majority), white cells and platelets (the minority) and it may be contaminated with a small amount of incompletely separated plasma. Thus SDRC is actually largely a mixture of red and white cells, though hereinafter, is referred to as the red cell fraction or SDRC. Since in cattle there is no detectable infectivity in blood there would be no infectivity in either the plasma or the cellular fraction. However, because TSE infectivity is strongly associated with PrP Sc and this is derived from a cell membrane protein PrP C if present it is most likely to be associated with the cellular fraction though some may also be free in the plasma. Human blood derived from healthy donors who at the time of donation were incubating vcjd has been incriminated in the transmission of vcjd infectivity to four recipients (three of which developed vcjd) by transfusion of red cells. In order to make an estimate of the risks resulting from the use of different fractions of human blood, risk assessments have been completed. For example, the level of infectivity in red cells is variously estimated (DNV, 2003) to be from 13%-68% and in buffy coat from 22%- 87% of that in whole blood. Thus if a similar situation occurred in cattle the proportion of the total infectivity that would be in the final SDRC could be considerable. It would also be impossible with current technology to remove or inactivate it and still retain the nutritional value of the product. Thus there is requirement to establish the safety of SDRC by selecting only safe sources as defined in this document (which is done by EAPA Member companies). Filter washing and waste disposal The precipitate collected on filters will also likely carry some infectivity if any was present in the raw material leaving the slaughterhouse. The filters are washed several times daily with running fresh water. The water with the accompanying debris is dealt with in one of two ways depending on the plant. In some EAPA members plants for example, there are an integral waste treatment procedure wherein the water is chemically and biologically treated. After treatment the water is discharged to the river and the solid waste is collected by a licensed waste removal firm for disposal in a licensed landfill site. Other plants may use similar systems or alternatively the waste water is discharged to a separate cleansing and waste treatment centre. Each procedure is authorized and supervised by the Competent Veterinary Authority and with the full agreement and knowledge of the local water authority and landfill owner. Thus waste material is disposed off in a safe and environmentally-friendly way and under official control. The CCP that theoretically might contribute to inactivation, as distinct from agent removal, is the spray-drying process itself. This will now be discussed in relation to the BSE agent. 26

72 BRIEF RISK ANALYSIS FOR SDRC THE HAZARD The hazard is from TSE agents from cattle, certainly the BSE agent as described by Bruce et al, (1997) and potentially from the agents that cause atypical TSE as described by Brown et al, (2006) and Buschmann et al, (2006) if different. The hazard is only likely to create a risk as a result of exposure by the oral route by consumption of feed containing the hazard. THE STARTING MATERIAL AND END PRODUCT The starting material is bovine blood as defined below. The end product is SDRC derived from the starting material or fish feed containing SDRC. The risk assessment will consider three factors: the SOURCE of the starting material, the PROCESS to which the starting material is subjected, and the USE to which it is put. SOURCE Species and risk The starting material, bovine blood, is derived only from domestic cattle of the species Bos taurus. An occasional source from cattle of the species Bos indicus cannot be entirely excluded. Both species are susceptible to natural BSE and for practical purposes can be assumed to present a similar risk. Geographical source and risk All cattle will be resident within the EU and will come largely from the following countries: Belgium, Denmark, France, Germany, Italy, Poland, Spain, Sweden, the Netherlands and the United Kingdom where EAPA member companies have processing plants. Because the EU is a free-trading area it cannot be excluded that cattle could come from other EU countries. For practical purposes all Member States of the EU have the same level of risk: in 2007 namely EFSA Category III (BSE likely but not confirmed or confirmed at a lower level) and an OIE Category of Controlled risk. It is noted that this is a judgement based on current knowledge and not an official categorisation simply because many countries have not had a recent official scrutiny (EFSA) or have not been classified into the OIE three category system (OIE). The TSE agent strain risk The only known naturally occurring TSE of cattle are classical BSE and atypical BSE. It seems likely but is not yet proven that these disease phenotypes are caused by TSE agents with different biological properties, i.e., different agent 27

73 strains. There is some evidence quoted by Brown et al, (2006) that in one form of transgenic humanised mice, one type of atypical BSE (BASE L type atypical BSE) transmitted whereas classical BSE did not, suggesting that it is possible that at least BASE cases may be more pathogenic for humans than is classical BSE that has one major strain (Bruce et al, 1997). Epidemiologically there is scant support for the notion that atypical BSE is a human health risk at all but Western blots of one BASE case show a similarity to that in humans with sporadic CJD and type 2 PrP Sc that predominates in patients with the MV polymorphism at codon 129 of the PRNP gene. Similarly one H type atypical BSE has a Western blot profile similar to that in humans with sporadic CJD and type 1 PrP Sc that predominates in patients with the MM polymorphism at codon 129 of the PRNP gene. As yet there has been no strong evidence to support a causal connection between cattle with atypical BSE and any form of human TSE. On the other hand there is strong support for the fact that classical BSE agent is responsible for human vcjd contracted by consumption of BSEinfected cattle products (SRM or uninfected tissues cross-contaminated with SRM). Furthermore the biological and molecular strains of agent from cattle and humans are closely similar. TSE risk in the tissue (bovine blood) No detectable infectivity has been found in bovine blood or blood components following bioassay in susceptible mice by the i/c and i/p route or cattle by the i/c route (see previous section for details and the WHO classification of bovine blood). There is no formal report that an infectious form of PrP Sc is present in bovine blood though some commercial companies have claimed they can distinguish blood from BSE infected cattle and control cattle. However, in the absence of detectable infectivity even following bioassay in cattle by the i/c route this is difficult to believe. Nevertheless this is why there is a disputed result in the WHO Table 1b for PrP presence. TSE risk resulting from cross-contamination The risk of cross-contamination of bovine blood with TSE infectivity in 2007 is extremely low because very few BSE infections are found in slaughter cattle of any age and especially in young cattle that form the bulk of animals for a slaughter batch. Furthermore, all cattle >30 months old are tested for BSE and if they fail the blood and all tissues (including blood) from that animal, the one before and two after in the slaughter line are destroyed and cannot be used for any purpose. If the blood has been batched from several animals the whole batch is destroyed. Thus it is highly unlikely that any bovine animal with infectivity in the brain could supply blood for making SDRC. Cross-contamination as a result of the use of particular stunning methods or pithing is also highly unlikely for the same reason that a positive BSE animal would be rejected. In addition, risk methods of stunning and pithing cattle are prohibited throughout the EU. Finally, crosscontamination from a rapid test negative animal is just as unlikely but any TSE risk would be lower still because the brain would not be infected. Exposure of 28

74 blood to other SRM from any animal is not possible because of the segregation of bleeding from carcase splitting and evisceration. The TSE risk from additions to the blood The only addition to the blood is anticoagulant which are inorganic salts not derived from animals. There is no hazard and therefore no risk from this material. Processing bovine blood to make SDRC The process used to manufacture SDRC is described elsewhere in this document. No part of the process is capable of inactivating TSE agents. However, various filtration steps remove blood clots and any large particles that inadvertently may rarely be present. Centrifugation separates the blood into a plasma fraction and a cellular fraction. The latter consists mostly of red cells, and volumetrically fewer white cells and platelets and a small amount of residual plasma. Since BSE infection has never been found in cattle blood it is not possible to determine where any infectivity (if present) resides. It is likely, based on studies in other species, to be mostly associated with white cells, particularly lymphocytes but it cannot be excluded that it is present in the other components. However, any infectivity present in plasma would likely remain there. The main point here is that the source is devoid of infectivity so the precise location of infectivity and the amount present is speculative. The process co-mingles blood from multiple animals and sometimes from multiple slaughterhouses. This inevitably results in mixing the blood from different sources and thereby it highly unlikely that if one animal actually contributed infected blood it would remain as one large packet in the SDRC but rather would be diluted by the blood or SDRC from the other animals in the batch. It is noted that at least four humans have been infected with vcjd following transfusion of non-leuco-depleted red cells from human blood donors that were incubating vcjd at the time of donation. The i/v route used for transfusion (that enable large volumes to be given) is about 1/10 as efficient as the i/c route whereas the oral route is the least efficient of all being approximately 500,000 less efficient than the i/c route. For all the reasons set out in this document any infectivity that is in bovine blood but is undetected, is bound to have a low titre, insufficient to contribute an infectious dose via feed. It can be concluded that processing blood to make SDRC would have very little, if any, effect of reducing the TSE infectivity should it be present in bovine blood. The use of SDRC The purpose of this document is to make the case for the use of bovine SDRC in the feed of farmed fish so only use in fish will be considered. Fish feed may 29

75 be pelleted and so if any SDRC used in the final pellet will be mixed with other materials. Assuming as determined above, that if there is no infectivity in the blood at the point of collection there could be none at the point of receipt of sealed bags at the compounders premises and the zero risk would remain. However, since SDRC would be used as an ingredient of farmed fish feed and this uses other animal derived products, experience informs us that the final ration could become contaminated with the BSE agent from other sources (e.g. infected MBM) by cross contamination. It is incumbent upon wholesalers, retailers, feed compounders and farmers to take the necessary precautions with storage, transport, feed preparation and mixing rations to reduce or eliminate risks from such sources that there may be. What is sure is that if there is a TSE source in the final feed it will be most unlikely to arise from the SDRC. Nevertheless it is important (and the EAPA support the notion) that any cycle of feeding must maximise the natural effects of the species barrier to reduce any risk, even negligible risks, to the lowest achievable in a commercial environment. To protect all species from exposure to TSE infectivity via feed the EAPA respects the need to apply tests especially to the commercial concentrate feeds for ruminant animals to ensure that prohibited ruminant protein, (including SDRC) is not present. Tests to achieve this are already available and used. CONCLUSION The TSE risk in SDRC derived from healthy EU cattle passed fit for human consumption is negligible. 30

76 ENVIRONMENTAL ISSUES RESULTING FROM DISPOSAL OF UNWANTED OR UNSAFE ANIMAL PRODUCTS General Since time immemorial there has been a need to dispose of animal material. Four main methods have been available since the XIX th century: consumption, burial, incineration and rendering. Of these only incineration at high temperature is accepted to be effective at destroying TSE agent infectivity. In the XXI st century even more methods are available including hydrolysis using chemicals and heat. Of the available methods consumption is the simplest and most effective but demands that the source is safe in respect of micro-organisms (including TSE agents) and toxins. Clearly, if the waste is unsafe there is a greater restriction on the method of disposal and the method chosen must itself be shown to be safe. The advent of BSE resulted in a massive increase in ruminant waste that previously had been used for consumption by man and animals. This waste is mostly in the form of carcases of infected and dead ruminant animals, SRM and other ruminant products that previously were permitted for various uses (including feed) but now have been prohibited for use either completely or partially. Bovine blood comes into this last category. Bovine blood Currently if the bovine blood from healthy slaughter animals is not collected for a specific use under the law, it becomes a Category 3 waste product (EC, 2002) that has to be removed from the abattoir for destruction by processing by an approved method followed by co-incineration, incineration or burial in licensed landfill. Such procedures result in added costs to the livestock and slaughter industry and inevitably put the EU livestock industry at a disadvantage in the world market. This view is supported by the European Economic and Social Committee (EESC) (EESC, 2006). The absence of SDRC in the diet of farmed fish means that the high protein content delivered by this material has to be obtained from other sources such as fishmeal and fish oil and soya protein for which there is competition. This adds further economic costs to the animal feed industry and ultimately to the consumer. There is an urgent need to reduce this dependency by finding other sustainable resources where production can keep pace with the growth of aquaculture which is increasing in the EU. One way to do this, and probably is the most economical available, would be to permit the use of bovine SDRC back into the diet of farmed fish. Organic potential contamination of blood as biological material According to Ruiz et al, (1993) and Tritt and Schuchardt (1992) the Chemical Oxygen Demand (COD) of whole blood is 0.4 kg O 2 / kg of blood. If we consider that the Organic Matter (OM) of the blood is 2/3 COD, this means that one kg of blood contains 0.27 kg of O 2 of OM. 31

77 If we assume that in developed countries a single person has a contamination impact of 70 g of OM / day, 1 kg of blood has the same potential contamination impact as 3.8 human individuals/day if it is not collected and treated appropriately. According to this evaluation, a plant that processes 75,000 metric tons of whole bovine blood per year, (340,000 kg/day) has an equivalent contamination impact per day to a city of 1,250,000 inhabitants. Maximizing the value of blood and adding value The use of SDRC not only maximises the value of the source product, it adds value and reduces the need for costly destruction. This view is in line with the view expressed by the EESC (EESC, 2006) and with the TSE Roadmap (EC, 2005a). FINAL CONCLUSION Blood from clinically healthy animals is an inherently safe product. If the risk management procedures recommended in this analysis are rigorously followed, any TSE risk for farmed fish from spray-dried bovine red cells in fish feed, is negligible. REQUEST The EAPA hereby request that urgent consideration is given to the reintroduction of SDRC into the diet of farmed fish by taking account of the current BSE situation in cattle in the EU, the safety of the starting material and end-product as determined in this document, the absence of TSE in fish and non-ruminant animals, the Regulations in place to protect humans and food animals from exposure to TSE agents and the improved environmental consequences of doing so. 32

78 REFERENCES ANIL MH, LOVE S, WILLIAMS S, SHAND A, McKINSTRY JL, HELPS CR, WATERMAN-PEARSON A, SEGHATCHIAN J, HARBOUR DA, (1999). Potential contamination of beef carcases with brain tissue at slaughter. Veterinary Record; 145: BRADLEY R, (1999). BSE transmission studies with particular reference to blood. Dev. Biol. Stand.; 99: BROWN P, McSHANE LM, ZANUSSO G, DETWILER L, (2006). On the question of sporadic or atypical BSE and CJD. Emerg Inf Dis, 12, BRUCE ME, WILL, RG, IRONSIDE JW, MCCONNELL I, DRUMMOND D, SUTTLE A, McCARDLE L, CHREE A, HOPE J, BIRKETT C, COUSENS S, FRASER H, BOSTOCK CJ, (1997). Transmissions to mice indicate that new variant CJD is caused by the BSE agent. Nature, 389, BUSCHMANN A, GRETZSCHEL A, BIACABE A-G, SCHIEBEL K, CORONA C, HOFFMANN C, EIDEN M, BARON T, CASALONE C, GROSCHUP MH, (2006). Atypical BSE in Germany - proof of transmissibility and biochemical characterisation. Vet. Microbiol, 117, BUSCHMANN A, GROSCHUP MH, (2005). Highly bovine spongiform encephalopathy-sensitive transgenic mice confirm the essential restriction of infectivity to the nervous system in clinically diseased cattle. J Inf Dis, 192, CASALONE C, ZANUSSO G, ACUTIS P, FERRARI S, CAPUCCI L, TAGLIAVINI F, MONACO S, CARAMELLI M, (2004). Identification of a second bovine amyloidotic spongiform encephalopathy: molecular similarities with sporadic CJD. PNAS, 101, COORE RR, LOVE S, McKINSTRY JL, WEAVER HR, PHILIPS A, HILLMAN T, HILES M, HELPS CR, ANIL MH (2005). Brain tissue fragments in jugular vein blood of cattle stunned by use of penetrating or non-penetrating captive bolt guns. J Fd Prot; 68, DALY DJ, PRENDERGAST DM, SHERIDAN JJ, BLAIR IS, McDOWELL DA, Use of a marker organism to model the spread of central nervous tissue in cattle and the abattoir environment during commercial stunning and carcass dressing. Appl Environ Microbiol, 68, DNV, Det Norske Veritas Consulting risk assessment of CJD in blood products for the UK Department of Health. Appendix II. Infectivity in blood. EC, (2000). Council Decision 2000/766/EC of 4 December 2000 concerning protection measures with regard to TSE and the feeding of animal protein. Off J EC, L 306,

79 EC, (2001a). Commission Decision 2001/9/EC of 29 December 2000 concerning control measures required for the implementation of Council Decision 2000/766/EC. Off J EC, L 2, EC, (2001b). Regulation (EC) No 999/2001 of the European Parliament and of the Council of 22 May 2001 laying down the rules for the prevention, control and eradication of certain TSE. Off J EC, L 147, 1-40 EC, (2001c). Commission Regulation (EC) No 1326/2001 of 29 June 2001 laying down transitional measures to permit the changeover to the Regulation of the European Parliament and of the Council (EC) No 999/2001 laying down rules for the prevention, control and eradication of certain transmissible spongiform encephalopathies, and amending Annexes VII and XI to that Regulation. Off J EC, L 177, EC, (2002). Regulation (EC) No 1774/2002 of the European Parliament and of the Council of 3 October 2002 laying down health rules concerning animal byproducts not intended for human consumption. Off J EC, L 273, EC, (2003a). Commission Regulation (EC) No 1234/2003 of 10 July 2003 amending Regulation (EC) No 999/2001 and Regulation (EC)1326/2001 as regards TSE and animal feeding. Off J EC, L 173, EC, (2003b). Commission Directive 2003/126/EC of 23 December 2003 on the analytical method for the determination of constituents of animal origin for the officialcontrol of feedingstuffs. Off J EU, L 339, EC, (2005a). The TSE Road Map. 15 July EC, Brussels. COM 322 FINAL. EC, (2005b).Commission Regulation (EC) No 1292/2005 of 5 August 2005 amending Annex IV to Regulation (EC) 999/2001 of the European Parliament and of the Council as regards animal nutrition. Off J EC, L205, EC, (2006). Report on the monitoring and testing of ruminants for the presence of transmissible spongiform encephalopathy (TSE) in the EU in June Directorate General for Health and Consumer Protection. EC, Brussels. EESC, (2006). Opinion on the disposal of animal carcasses and the use of animal by-products. 23/12/2006. Off J EU, C318, EFSA (2006) Geographical BSE Risk (GBR) assessments covering List of countries and their GBR level of risk as assessed by the Scientific Steering Committee and the European Food Safety Authority. TSE/BSE Risk Assessment Team of the Scientific Panel on Biological Hazards. 1 August essments/summary_list_countries.par.0001.file.dat/gbr_assessments_table_ Overview_assessed_countries_ pdf 34

80 EFSA (2007). Opinion of the Scientific Panel on Biological Hazards on the assessment of the health risks of feeding of ruminants with fishmeal in relation to the risk of TSE. Question N EFSA-Q Adopted on 24 January EFSA J, 443, 1-26 EUROPEAN PARLIAMENT (2004). Working Paper Fish 113 En. The fish meal and fish oil industry. Its role in the Common Fisheries Policy. Directorate- General for Research Fisheries Series. 0meal%20oil FRASER H, FOSTER JG, (1994). Transmission to mice, sheep and goats and bioassay of bovine tissues; in Bradley R, Marchant B (eds): Transmissible Spongiform Encephalopathies. Proceedings of a Consultation with the Scientific Veterinary Committee of the CEC, Sept VI/4131/94-EN Brussels, EC, pp GARLAND T, BAUER N, BAILEY M, (1996). Brain emboli in the lungs of cattle after stunning. Lancet; 348: 610. HOUSTON F, FOSTER JD, CHONG A, HUNTER N, BOSTOCK CJ. (2000). Transmission of BSE by blood transfusion in sheep. Research letter. The Lancet. 356: HUNTER N, FOSTER J, CHEONG A, McCUCHEON S, PARNHAM D, EATON S, MacKENZIE C, HOUSTON F Transmission of prion diseases by blood transfusion. J Gen Virol, 83, INGROSSO L, NOVOA B, DALLA VALLE AZ, CARDONE F, ARANGUREN R, SBRICCOLI M et al, (2006). Scrapie infectivity is quickly cleared in tissues of orally-infected farmed fish. BMC Vet Res, 2, KIMBERLIN RH, WILESMITH JW, (1994). Bovine spongiform encephalopathy. Epidemiology, low dose exposure and risks. Annals of the New York Academy of Sciences; 724: MHS (1998). Meat Hygiene Service: Annual report and Accounts, The Stationery Office, London. Pp.82. MUNRO R, (1997). Neural tissue embolism in cattle. Veterinary Record; 140: 536. OIE, (2006). Terrestrial Animal Health Code. Chapter Bovine Spongiform Encephalopathy. OIE Paris. PELEG M, COLE MB, (1998). Reinterpretation of Microbial Survival Curves. Clinical Reviews in Food Science. 38:

81 RUIZ I, VERGA MC, De SANTIAGO P, BLÁZQUEZ R. (1993). Características de los efluentes de matadero. Alimentación, equipos y tecnología. Septiembre- 93.: SCHMIDT GR, HOSSNER KL, YEMM RS, GOULD DH, (1999). Potential for disruption of central nervous system tissue in beef cattle by different types of captive bolt stunners. Journal of Food Protection; 62: SSC (1997). Scientific Opinion of the Scientific Steering Committee of the EC on the Listing of specified risk materials: a scheme for assessing relative risks to man. Adopted 9 December EC, DG XXIV, Brussels. Pp 20. SSC, (1999). Opinion of the Scientific Steering Committee (SSC) of 17 September 1999 that there is no evidence of the natural occurrence of TSE in non-ruminant farmed animals producing food such as pigs and poultry SSC (2000). Scientific Opinion of the Scientific Steering Committee of the EC on the implications of the Houston et al. Paper in The Lancet of 16 September 2000 on the transmission of BSE by blood transfussion in sheep. (The Lancet, Vol 356, pp ; ; 1013). Adopted October EC Health & Consumer Protection Directorate General. Pp 12. TRITT WP, SCHUCHARDT F. (1992). Materials flow and possibilities of treating liquid and solid waster from slaughterhouses in Germany. A review. Bioresource Technology 41: WELLS GAH, SCOTT AC, JOHNSON CT, GUNNING RF, HANCOCK RD, JEFFREY M, DAWSON M, BRADLEY R, (1987). A novel progressive spongiform encephalopathy in cattle. Vet Rec, 121, WELLS GAH, Tissue infectivity/prp BSE distribution and pathogenesis in bovine spongiform encephalopathy (BSE). Presentation at 10 th Annual Transmissible Spongiform Encephalopathies The Definitive American TSE Meeting, 7-9 March Cambridge Healthtech Institute, Baltimore. WHO, (2006). WHO Guidelines on tissue infectivity distribution in TSE. WHO. Geneva, Pp 53. WILESMITH JW, WELLS GAH, CRANWELL MP, RYAN JBM (1988). Bovine spongiform encephalopathy: epidemiological studies. Vet Rec, 123,

82 ANNEXES 37

83 Annex 1 EAPA, EUROPEAN ANIMAL PROTEIN ASSOCIATION EAPA is the European Animal Protein Association. It was founded in 1988 to represent companies that specialise in the production and supply of high quality natural blood proteins. These are valued ingredients in food products, in feeds for farm animals and pets and in aquaculture feeds. They also fulfil important roles in many pharmaceutical products. Blood proteins offer an excellent way of retaining the value from by-products of the meat processing industry that otherwise would be treated as less valuable commodities, or waste. EAPA represents all the blood protein producers in Europe. Membership includes major producers in Belgium, Denmark, France, Germany, Italy, Poland, Spain, Sweden, the Netherlands and the United Kingdom. Through membership of the association these producers have formed a network that enables technical information and scientific data to be shared with the objective of maintaining the highest standards of quality and safety in their end-products. This in turn enables the highest achievable safety and nutritional quality to be attained and developed in human food, animal and fish feed and pharmaceutical products that contain company products. The secretariat of EAPA is in Brussels, where close contact can be maintained with the European Commission, helping to ensure blood protein products continue to meet all legislative and advisory requirements with respect to quality and safety. EAPA contact information: European Animal Protein Association Boulevard Baudouin 18, 4th floor, BE Brussels Belgium Tel +32 (0) Fax +32 (0) info@eapa.biz Further information is available on the EAPA website: 38

84 Annex 2 ASSURANCE PROCEDURES TO GUARANTEE THE SAFETY OF SPRAY-DRIED BOVINE RED CELLS We believe that the ban introduced by the Council (Council Decision 2000/766/EC) and maintained by the Regulation 999/2001/EC is a precautionary measure on a temporary basis. We appreciate the need to break the chain of possible TSE infection that may have been entering ruminant feed by cross contamination in some Member States. We also believe that Expert Group Reports, the EFSA and the European Commission have no scientific grounds for prohibiting the use of spray dried bovine red cells or bovine blood products (as specified in this document) in farmed fish feed on the grounds of TSE risk. This document independently reviews the TSE risk in spray dried bovine red cells for farmed fish feed and the paragraphs below provide the essential information on collection procedures, processing and use, that will give sound guarantees for decision makers to be satisfied that a TSE risk is excluded if spray dried bovine red cells are again permitted into farmed fish feed. Finally, because there is sound scientific support for a negligible TSE risk in bovine blood products both now and historically, and because the Commission Regulation intends only a temporary ban, as also indicated in The TSE Road Map (COM, 2005) point 2.2 that The starting point when revising the current feed ban provisions should be risk-based as documented in the present report, therefore we urge that consideration be given to lifting the ban as soon as possible. Control at the collection site Cattle slaughterhouses (EU Member States) Blood is collected only from healthy cattle that have passed an ante and postmortem inspection following veterinary inspection. Blood is collected only from cattle less than 30 months old or, if over 30 months old, the said cattle must pass an approved EU rapid test for the detection of abnormal prion protein in the CNS. Only EC-approved stunning methods are used and pithing is prohibited. Additionally all the slaughterhouses used by EAPA members provide a certificate stating that the place of slaughter is physically separate from the place of carcase splitting and evisceration. This eliminates the TSE risk from SRM other than via the stun hole or method of stunning 39

85 EAPA member s equipment used to collect blood is exclusively dedicated to the collection of bovine blood. The closed circuit from the point of collection to the storage tank in the slaughterhouse avoids potential contamination after the collection of the blood. Control during transportation Exclusively dedicated isothermal trucks with driver certificate that there is no manipulation of the load from the point of collection to the plant. Each load comes with a commercial document, as requested by Regulation (EC) Nº 1774/2002, from the slaughterhouse where quantity, slaughterhouse identification, blood temperature and date of collection are registered. Control during delivery in the plant Date, time, driver, truck identification number, quantity of blood delivered, destination tank in the manufacturing plant, slaughterhouse and Quality Control parameters (temperature, colour, etc.) are registered and controlled prior to unloading in the manufacturing plant. Before unloading a trained plant worker verifies and signs that the incoming blood is coming exclusively from the list of slaughterhouses that have been approved for collection of blood. Control at the manufacturing plant The manufacturing process takes place in a closed system to eliminate the risk of contamination. The product is bagged in new bags properly identified. Each bag is identified with a lot number code that identifies the manufacturing plant, date of production, production line, expiry date and production shift. The pallets of bagged products contain information about product identification, lot number. Control of final product Quality control Each batch of spray dried bovine blood derivatives is tested for physicochemical and microbiological analysis. All the lots are analysed for the presence of Salmonella species and Enterobacteriaceae. Each batch sold must have no Salmonella species present in 25 g. and less than 300 cfu/g for Enterobacteriaceae and conforms to the criteria laid down in Chapter I, paragraph 10 of Annex VII of Regulation (EC) Nº 1774/

86 If any lot (usually the amount of SDRC produced from one day s blood collection), fails any test the lot is withdrawn from sale for use in animal feed and it is disposed of in a safe manner in accordance with the law. This would most likely be by incineration or burial at a licensed landfill site. The EAPA members have established a safety measure that ensures the retention of final product in the warehouse, at least for 3 days before delivering the product to the final customer. This measure allows time enough to perform all the analysis and also guarantees an extra time for the competent authority to identify any infectious disease hazard detected within the 5 days after the sacrifice of the animals. Customer complaints of any kind are thoroughly investigated. During the last 15 years not a single case has revealed the implication in any bacteriological, viral or fungal diseases due to the use of spray dried blood derivatives. (J. Polo, personal communication). Traceability Every single kg of product manufactured in EAPA member s plants is assigned a production lot number that identifies the manufacturing plant, date of production, production line, expiration date and production shift. Every single lot is checked for physico-chemical and microbiological analysis. Our system allows us to document the quality of every single lot (warehouse, quarantine or available for sale). We also document every lot sold to the customer, date of delivery and the quantity of product sent to each customer. This information allows EAPA members to trace any lot number from the source of incoming blood (collection site or slaughterhouse). Traceability of incoming blood also allows identification of the farm of origin and destination of the final product (customer) in hours. In the hypothetical situation of a potential hazard this permits EAPA member-s to react immediately, immobilising the product in the facilities, and informing the customer if the product has already been delivered. 41

87 Annex 3 QUALITY PROCEDURES: HACCP, ISO 9000 EAPA member-s plants are working under strict quality procedures such as HACCP, ISO-9000 or equivalent quality rules. The EAPA and individual EAPA Member Companies have collaboration agreements with universities, research centres and laboratories conducting official tests in order to compare analytical methods, to test random samples or to investigate unusual events. Direct contact with national and other regulatory agencies, official veterinarians, universities and consultants ensures that EAPA members are regularly and promptly informed about new hazards, changes in legislation or any new requirements. 42

88 Annex 4 A DESCRIPTION OF THE PROCESS TO PREPARE BOVINE SDRC (as prepared by the EAPA) Source animals Cattle s arrive at the slaughterhouse from surrounding farms (FIGURE 1) and are held in the lairage until a clinical, ante mortem inspection is completed under the supervision of the Official Veterinary Surgeon (OVS). Animals showing clinical signs of disease may be held in the lairage for further observation, may be returned to the farm whence they have come, or be sent to an appropriate place for slaughter and destruction. FIGURE 1 Basic inspection, slaughter and test procedures LIVE, HEALTHY CATTLE LICENSED SLAUGHTER HOUSE WITH OFFICIAL VETERINARY SURGEON SUPERVISION ANTE MORTEM INSPECTION PASS: - Slaughter - Collect blood FAIL: - Isolate or - Return to farm or - Slaughter and destroy elsewhere POST MORTEM INSPECTION If > 30 months old Rapid test FAIL - Destroy blood batch PASS - Blood for SDRC Only animals that have passed the ante mortem inspection are permitted to be slaughtered. EAPA members only collect blood from such animals. Furthermore, blood is only collected for processing into SDRC if it is derived from carcases passed as fit for human consumption in accordance with Community legislation (i.e. after an official post mortem inspection) and is not affected by any signs of diseases communicable to humans or animals. 43

89 It is noted that it is incumbent on the Competent Veterinary Authority to determine that animals for slaughter come from regions, zones and farms devoid of notifiable diseases. Slaughter and collection of blood After the administrative procedures like animal identification (and weighing if appropriate) animals proceed to slaughter. For welfare, safety and other reasons, animals are stunned before killing. Exceptions are for religious (Kosher and Halal) slaughter of cattle where killing is done by cutting the throat. Cattle are usually stunned using a captive bolt pistol (MHS, 1998). This takes place in a restraining knocking box that enables the slaughterman to achieve an accurate hit to the appropriate part of the skull. Such a bolt makes a hole in the skull and penetrates the brain but there are some non-penetrative percussion pistols in use. The captive bolt may be powered by a cartridge or by compressed air. See page 22 for information on TSE risks related to stunning using methods injecting compressed air or gas into the cranial cavity.and pithing. This is no longer relevant in the EU as these risk methods have been banned by EC Regulation 999/2001 (EC, 2001). It is noted that captive bolt pistol bolts and sticking knives may be washed and cleaned in hot water after use but not to the standards necessary to destroy TSE agents. Following hoisting, the stunned beast is stuck with a sticking knife at the lower end of the neck, a process that severs the major blood vessels including at least one of the carotid arteries and jugular veins. Blood gushes from the stick hole to drain directly through a plastic tube incorporated in the knife into a tank (hollow knife system), or from the stick hole to a canal or collection trough that receives the blood from multiple animals. For the collection of blood for the preparation of bovine SDRC it should not be allowed to clot. To prevent this, blood is mixed with a solution of sodium citrate and/or sodium phosphate. Collections can be made singly but this is generally not practical for large numbers of animals. Thus it is usually pooled at the point of collection. Risks of TSE contamination from other specified bovine offal such as the spinal cord or intestine, is most unlikely because these tissues are only exposed much later in the slaughter hall and well away from the blood collection point The blood is filtered at the slaughterhouse and at the manufacturing plant. Following filtration, the blood is collected in a refrigerated stainless steel storage tank and chilled to 4 C. The storage tank co-mingles blood from multiple animals. One tank may hold the blood from adult cattle. Transportation Blood is pumped from the refrigerated tanks using a closed system to dedicated road tankers. It is transported to the manufacturing plant where it is filtered 44

90 again twice and centrifuged. The two processes also assist in removing gross particles. Following centrifugation there are three further filtration steps. For details of filtration and the mesh sizes used see the section on Risk Analysis Process. Immediately following centrifugation the plasma and red cell fractions are transferred to separate refrigerated, stainless steel tanks and held at 4ºC. All plants are dedicated to only processing blood from a single species and operate to international standards such as ISO 9000 using HACCP or equivalent quality rules. SPRAY DRYING THE RED CELL FRACTION The spray drying process is illustrated in FIGURE 2 and specific nozzles used in the process are shown in FIGURES 3 5. Spray drying is accomplished in a chamber. The process is as follows. Heated air. The air circulating through the drying chamber is atmospheric air, finely filtered and warmed by passing through a steam heater or an indirect gas heater. A centrifugal ventilator moves the heated air into the circulation system. The spray drying process used for the preparation of spray-dried red cells at EAPA member s facilities has an inlet temperature of at least 240ºC, range 240- to 270ºC (minimum contact time is 15 seconds but may be up to 30 seconds in other plants) and an outlet temperature of 80-90º C. Injection. The red cells are injected into the drying chamber, at some height above the floor, as very fine drops (10-200µm diameter) using a high-pressure nozzle. The type of nozzle used depends on the configuration of the drying chamber and flow of heated air. This allows a direct contact between the cell drops and the heated air stream. There is thus a high surface area to volume ratio that ensures rapid and efficient heating of the drops. This allows very fast drying of the product, which becomes a dry powder and falls to the floor by gravity. There are mathematical equations that correlate the temperature of the air and the exposure time of the product to these temperatures (Peleg and Cole, 1998; The time of residence of the particles in the drying chamber can be controlled and adjusted to the desired conditions of time and temperature. 45

91 FIGURE 2 SPRAY DRIED RED CELLS MANUFACTURING PROCESS SPRAY DRIED RED CELLS PRODUCTION PROCESS Blood processing The blood processing industry is analogous to the milk processing industry. Equipment, processing methods and quality control standards are similar in both industries. Dedicated stainless steel, food grade equipment is used. Filtration steps during processing For details see the section on Risk Analysis Processing. Spray drying Spray drying is widely used in the food industry. Examples of spray dried products are whole and skinned milk, butter and cheese, eggs, coffee and tea, cereal derivatives, fruit and vegetable juices, yeast extracts, meat extracts, edible proteins, baby food, dairy products, blood serum and plasma. Spray drying involves injecting a product at high pressure into a heated drying chamber to form very fine droplets. The drying chamber is the part of the system where the tiny red cells droplets contact the heated air and the drying process takes place. There are different kinds of drying chambers adapted to the spray drying of different products. The droplets encounter a stream of heated air that quickly evaporates Plasma moisture to form a dry powder. It is important that the drops are homogeneously Fraction sized and arrive at a consistent rate so that all the particles are exposed to the same temperature conditions. Specially designed and engineered nozzles are used to achieve this. Red Cells Fraction Nozzles. There are three main kinds of nozzle. The pressure nozzle (FIGURE 3) uses high pressure to spray the material into the heating chamber. The centrifugal nozzle (FIGURE 4) sprays red cells via a high speed centrifuge disk. The two fluid nozzle (FIGURE 5) sprays the cells with the energy of a high speed air stream. 46

92 FIGURE 3: Pressure Nozzle. Pressure N ozzle (by Niro Atom izer Ltd., Denm ark) FIGURE 4: Centrifuge Nozzle FIGURE 5: Two-Fuid Nozzle Nozzle Centrifuge Head (by Niro Atomizer Ltd., Denmark) 2 Fluid Nozzle Operation Principle (by Niro Atomizer Ltd., Denmark) B DEHYDRATATION A Axis of Rotation B Feeding Tube C Radial Canal D Dispenser The main components of the Spray Dryer are shown in FIGURE 6. FIGURE 6: Spray-Drying System Typical drying process by atomization (by Niro Atomizer Ltd., Denmark) Feeding Tank 2 Fedding Pump 3 Feeding Tube 4 Atomizer 1 3-steps Valve 7 Air Filter 8 Air Intake 9 Air Heater 11 Air Dispersion 12 Drying Chamber 13 Feeding Shaker Pneumatic Transport System 16 Clicon 17 Ventilation Dumping 18 Stack 19 Ciclon 21 Rotary Valve 22 Ventialtion for Air Transport 23 Air Filter 24 Instrument Board 5 Water Tank 10 Air Duct 15 Outler Pipe 20 Hopper 25 Ventilation for cooiing the Atomizer Air