Prion Infected Meat-and-Bone Meal Is Still Infectious after Biodiesel Production
Cathrin E. Bruederle1*, Robert M. Hnasko1, Thomas Kraemer2, Rafael A. Garcia3, Michael J. Haas3, William N. Marmer3, John Mark Carter1
1 USDA-ARS WRRC, Foodborne Contaminants Research Unit, Albany, California, United States of America2 Forensic Toxicology, Institute of Legal Medicine, Saarland University, Homburg/Saar, Germany3 USDA-ARS ERRC, Fats, Oils and Animal Coproducts Research Unit, Wyndmoor, Pennsylvania, United States of America
Abstract The epidemic of bovine spongiform encephalopathy (BSE) has led to a world-wide drop in the market for beef by-products, such as Meat-and-Bone Meal (MBM), a fat-containing but mainly proteinaceaous product traditionally used as an animal feed supplement. While normal rendering is insufficient, the production of biodiesel from MBM has been suggested to destroy infectivity from transmissible spongiform encephalopathies (TSEs). In addition to producing fuel, this method simultaneously generates a nutritious solid residue. In our study we produced biodiesel from MBM under defined conditions using a modified form of alkaline methanolysis. We evaluated the presence of prion in the three resulting phases of the biodiesel reaction (Biodiesel, Glycerol and Solid Residue) in vitro and in vivo. Analysis of the reaction products from 263K scrapie infected MBM led to no detectable immunoreactivity by Western Blot. Importantly, and in contrast to the biochemical results the solid MBM residue from the reaction retained infectivity when tested in an animal bioassay. Histochemical analysis of hamster brains inoculated with the solid residue showed typical spongiform degeneration and vacuolation. Re-inoculation of these brains into a new cohort of hamsters led to onset of clinical scrapie symptoms within 75 days, suggesting that the specific infectivity of the prion protein was not changed during the biodiesel process. The biodiesel reaction cannot be considered a viable prion decontamination method for MBM, although we observed increased survival time of hamsters and reduced infectivity greater than 6 log orders in the solid MBM residue. Furthermore, results from our study compare for the first time prion detection by Western Blot versus an infectivity bioassay for analysis of biodiesel reaction products. We could show that biochemical analysis alone is insufficient for detection of prion infectivity after a biodiesel process.
Citation: Bruederle CE, Hnasko RM, Kraemer T, Garcia RA, Haas MJ, et al. (2008) Prion Infected Meat-and-Bone Meal Is Still Infectious after Biodiesel Production. PLoS ONE 3(8): e2969. doi:10.1371/journal.pone.0002969
Editor: Neil Mabbott, University of Edinburgh, United Kingdom
Received: April 21, 2008; Accepted: July 24, 2008; Published: August 13, 2008
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: CRIS 5325-32000-007-00D and CRIS 5325-32000-008-00D
Competing interests: The authors have declared that no competing interests exist.
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Discussion Decontamination of pathogenic prions has turned out to be a challenging endeavor. Prions are known to be unusually resistant to common decontamination methods. BSE is believed to be a result of insufficient decontamination and rendering methods of ruminant coproducts that were used as animal feed. Although this led to a devastating feed-borne epidemic among cattle, a major concern here is the overwhelming evidence for the zoonotic transmission of bovine prions to humans . Total elimination of TSEs requires methods that completely destroy any potential prion infectivity in a large scale format. Production of biodiesel from bovine fat and brain tissue has been proposed to be a useful tool for decontamination of prions resulting in safe biodiesel . In our study we evaluated an inexpensive large scale method (in situ transesterification) for production of biodiesel for TSE decontamination potential. Furthermore we investigated potential infectivity present not only in the biodiesel but also in the two other phases developed from the process, a solid MBM residue and glycerol. The solid MBM residue is of particular interest for its potential as a nutritious feed additive for ruminants such as cattle. In our hands, under optimal conditions for transesterification, the solid MBM residue retained 7% of the initial triglyceride and 90% of the initial protein content 
The alkaline methanolysis method efficiently produced biodiesel from MBM spiked with hamster brain and the method eliminated PrPsc detection in all products as determined by Western blot. Our biochemical results are comparable to previous studies, at least with regards to the biodiesel and glycerol phase . Biodiesel and glycerol products had no detectable infectivity in our long term animal assay (survival>200d). In contrast to the biodiesel and glycerol phase, we show that the remaining solid MBM residue that had been spiked with scrapie brain retained infectivity in our sensitive bioassay. All animals inoculated with the infected solid MBM residue developed scrapie. However, increased survival time suggests the reaction did reduce infectivity in solid MBM residue from 10-3 ID50 to 10-9 ID50 (a partial decontamination of ~6 logs), based on a standard hamster survival curve that we established in our laboratory according to previous reported results . The broad distribution of time-to-death for these animals is likely due to uneven distribution of infectious material in the inoculum, as the residue produced a relatively coarse suspension in the syringe. We suggest that, in addition to disinfection by the alkaline methanolysis reaction, we observe significant partitioning of infectivity, from the liquid phases into the solid residue. Another possible explanation for increased survival of animals inoculated with the solid MBM residue could be a high binding affinity of the prion protein to MBM and thus a sustained release from MBM in the brain. A phenomenon like this was described previously for prion binding to soil minerals . In our study, when spiked into MBM, PrPsc was only detectable by Western Blot after boiling of sample in detergent. On the other hand we could show that control animals that received infected MBM not subjected to the reaction (MBM sc) developed disease in a time frame comparable to a standard scrapie brain homogenate.
Our results clearly show that Western Blot detection alone is insufficient to conclude on the absence of infectious prion, particularly when assessing a grossly heterogeneous sample such as MBM. This study illustrates that lack of prion detection in vitro does not necessarily exclude infectivity as determined by bioassay.
Furthermore the residual scrapie infectivity detected in the solid MBM residue probably limits the use of ruminant MBM as a feed additive to only non-ruminants, such as fish and fowl, as they are not susceptible to TSEs. Relatively minor variations of this reaction (e.g., more heat and/or alkali) may prove fully effective for complete destruction of infectivity in the solid MBM residue, but must be cost-effective if suspect MBM is to be considered as a ruminant feed additive.
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