Conceived and designed the experiments: CEB RMH WNM JMC. Performed the experiments: CEB RMH TK. Analyzed the data: CEB TK JMC. Contributed reagents/materials/analysis tools: CEB RMH TK RAG MJH JMC. Wrote the paper: CEB.
The authors have declared that no competing interests exist.
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.
Bovine spongiform encephalopathy (BSE) belongs to the transmissible spongiform encephalopathies (TSEs), a family of fatal neurodegenerative diseases. Besides BSE in cows (‘mad cow disease’), other TSEs include Chronic Wasting Disease (CWD) in elk and deer, Creutzfeldt-Jacob Disease (CJD) in humans and scrapie in sheep.
The hallmark of TSEs is the conversion of cellular prion protein (PrPc) to an abnormal isoform (prion, PrPsc) relatively rich in beta sheet structure
Insolubility and protease resistance is used to distinguish PrPsc from PrPc biochemically. Limited proteolysis of PrPsc by proteinase K but not PrPc produces a smaller protease resistant residue of approximately 142 amino acids that can be detected by Western Blot
Unlike viruses, prions are infectious proteins and transmissibility is not dependent on nucleic acid
Another defining characteristic of TSEs is transmissibility between individuals or across species
Production of biodiesel from animal fat has been proposed to decontaminate any potential prion infectivity present in the biodiesel
In our study we produced biodiesel from MBM spiked with 263K scrapie, a model for TSE infectivity, using
We validated biodiesel production from MBM by gas chromatography and evaluated all three phases derived from the reaction (Biodiesel, Glycerol and Solid MBM Residue) for the presence of proteinase K resistant prion biochemically and prion infectivity by animal bioassay.
MBM entirely derived from cattle bones and offal was spiked with 5% brain homogenate from either normal or 263K scrapie infected hamsters, and then subjected to alkaline methanolysis. The reaction was performed by vigorous shaking of MBM in 0.25% sodium methoxide for 2 hours at 35°C. After settling of the mixture overnight we could distinguish 3 independent reaction phases. The upper phase, representing the potential biodiesel, was separated and analyzed by gas chromatography (
After alkaline methanolysis of MBM the upper biodiesel phase was separated and analyzed by GC-MS. GC-MS results show the alkaline methanolysis reaction produced various fatty acid methyl esters (FAME) which are typical components of biodiesel (see
1 | Tetradecanoic acid methyl ester |
2 | Pentadecanoic acid methyl ester |
3 | Hexadecenoic acid methyl ester |
4 | Hexadecanoic acid methyl ester |
5 | Heptadecanoic acid methyl ester |
6 | Octadecadienoic acid methyl ester |
7 | Octadecenoic acid methyl ester |
8 | Octadecanoic acid methyl ester |
Numbers indicate the spikes shown in
Fatty Acid Methyl Ester (FAME) | Percentage of total FAME produced |
Stearate (Octadecanoic acid methyl ester) | 20% |
Palmitate (Hexadecanoic acid methyl ester) | 17% |
calculated according to Haas et al.
In order to validate the infectivity of the three different resulting phases from the biodiesel reaction we needed to perform an
In order to investigate the decontamination potential of the biodiesel reaction under our defined conditions we performed alkaline methanolysis on MBM spiked with 5% 263K scrapie infected brain (MBM sc). After the reaction the three chemical phases were separated and each phase was subsequently diluted to an estimated amount of 25 µg of total protein into 50 µl of 320 mM sucrose solution to prepare a homogeneous sample for intracranial (
We prepared an identical sample set performing alkaline methanolysis on MBM c as negative control for
First we performed Western Blots on the crude fractions from the reaction as well as on the above described inocula (
We performed alkaline methanolysis on scrapie brain spiked MBM as described under
To confirm our results obtained by Western Blot we used a hamster bioassay to assess prion infectivity in all three reaction phases. Syrian hamsters were inoculated with biodiesel, glycerol or solid MBM residue produced from either MBM sc or uninfected MBM c (negative control). Furthermore, we inoculated untreated MBM c and MBM sc as pre-reaction controls. A group of hamsters inoculated with 1% scrapie brain homogenate served as additional control to prove the integrity of our animal assay (
(A) Control animals inoculated with 50 µl 1% scrapie BH exhibited clinical symptoms 75 days after inoculation and were sacrificed (1% sc brain). Animals inoculated with MBM sc began to display clinical symptoms around 75 days post inoculation and were sacrificed when moribund. All animals inoculated with Solid MBM Residue sc developed disease (166+/−13 days), even though no PrPsc was detected by Western Blot in initial inoculum. Survival time of animals from all 3 groups was significantly shorter as compared to control animals (control: MBM c and Solid MBM Residue c; p<0.05; ANOVA on ranks). Animals inoculated with Biodiesel sc and Glycerol sc displayed no clinical symptoms within the time frame of the experiment (>200 days). Survival was comparable to control group animals (Biodiesel c, Glycerol c). Days of survival are calculated as mean values. (B) The range of time-to death values of hamsters inoculated with MBM sc and Solid MBM Residue sc is summarized showing the median, upper and lower quartiles and extreme values (outliers) for comparison of both groups.
Inoculum | spiked with Infected Brain (Sacrificed Animals/Total animals) | Day Sacrificed |
Biodiesel phase | 4/14 |
|
Glycerol phase | 5/14 |
|
mean+/−standard error
unsacrificed animals survived longer than 200 days, until study was terminated, without clinical scrapie symptoms
sacrificed animals exhibited clinical scrapie symptoms
Most interestingly, all animals inoculated with the solid MBM residue produced by the reaction with scrapie brain spiked MBM developed disease (
No animals inoculated with biodiesel or glycerol fractions derived from infected MBM (Biodiesel sc, Glycerol sc) died with scrapie symptoms. The 28% loss of animals in these groups is comparable to previous reported attrition during long-term experiments. Secondary passage of brain homogenate from these animals was free of scrapie infectivity (survival>200 days). A minor loss of animals in the negative control groups was not due to scrapie infectivity, but rather to traumatic injuries from fighting (
After termination of the animal bioassay we performed Western Blot analysis of select brains from animals from the different treatment groups to determine the presence of proteinase K (PK) resistant material (
After the animal bioassay was terminated we evaluated brains from the different treatment groups for presence of PrPsc. We could detect PrPsc in brains from animals treated with infected solid MBM residue (4A, Solid Residue sc, +PK) as well as animals that received MBM spiked with infected brain (4A, MBM sc, +PK). Samples were digested with proteinase K to verify presence of PrPsc and detected with monoclonal (mAb) IPC1. Brain homogenates from inoculated biodiesel and glycerol fractions contained no PK resistant material whether derived from infected or uninfected MBM (4B). This suggests that death of animals inoculated with the solid MBM residue was definitely due to infection with scrapie whereas the biodiesel and glycerol phase of the reaction can be considered as decontaminated due to lack of any detectable protease resistant material in brains from animals injected with the respective phases.
These data show that the production of biodiesel and glycerol from prion contaminated MBM led to no detectable PrPsc or prion infectivity in the applied assays. In contrast, the solid MBM residue retained infectivity in the animal bioassay and cannot be considered as decontaminated and thus could still serve as a vector for disease transmission.
To confirm scrapie brain pathology from animals with clinical scrapie symptoms we evaluated brain sections counterstained with Hematoxylin-Congo red. Typical vacuolation of prion disease was observed in brain sections from animals inoculated with the Solid MBM Residue sc (
Coronal brain sections were stained with Hematoxylin-Congo red. (A, B) Control animals inoculated with the solid MBM residue resulting from the reaction with MBM+5% uninfected brain exhibited normal brain morphology without vacuolation or amyloidosis. (C, D) Cryo-sections of hamster brain stained with Hematoxylin-Congo red show vacuolation (arrows) when animals were inoculated with the Solid MBM residue resulting from the reaction with MBM+5% infected brain suggesting scrapie infection. Both animals were sacrificed 150 days post-inoculation, when the infected animal displayed severe scrapie symptoms.
Secondary passage of brain homogenates was conducted from animals in all reaction groups. Brain homogenates from animals that received Solid MBM Residue sc, MBM sc and Biodiesel sc and Glycerol sc were inoculated into new groups of hamsters to assess results from the initial bioassay. Animals that received inocula prepared from brains that were treated with Solid MBM Residue sc and MBM sc developed clinical scrapie symptoms at 70 days and were sacrificed at 80 days (data not shown). This confirms that MBM sc can transmit scrapie following alkaline methanolysis. Survival time after re-inoculation was comparable to the survival time after inoculation of a standard 1% scrapie brain homogenate (
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
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
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
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.
25 µl of the sample was dried under a constant stream of nitrogen. The dried residue was reconstituted in 1.0 ml of isooctane. 1 µl of this solution was injected into the GC-MS.
The samples were analyzed using a Hewlett Packard HP 6890 Series GC system combined with an HP 5973 Series mass selective detector, an HP 6890 Series injector and an HP Chem Station G1701AA version A.03.00. The MS conditions were as follows: full scan mode; electron impact ionization (EI) mode: ionization energy, 70 eV; ion source temperature, 220°C; capillary direct interface heated at 260°C.
GC conditions were as follows: split less injection mode; column, HP-1MS capillary (25 m_0.25 mm I.D.), 250 nm film thickness; injection port temperature, 280°C; carrier gas, helium; flow rate, 0.6 ml/min; column temperature, 100°C increased to 310°C at 30°C/min, and was held at this temperature for 5 min.
MBM samples were obtained through the Fats and Proteins Research Foundation (Alexandria, VA) from an anonymous member company. The supplier specified that the MBM was made entirely from cattle bones and offal. The material had about 43% protein and 37% ash.
We prepared small, representative samples from this very heterogeneous material. Initially a mortar and pestle was used to reduce the size of the largest pieces and break up large aggregates. Then the material was mixed well and split four times with a 10-slot riffle box, resulting in a ∼120 g representative sample. This material was milled under liquid nitrogen in a Spex Centriprep model 6800 mill (Spex Centiprep Inc., Metuchen, New Jersey, United States). Milling the material into such a fine powder does not represent industrial practice. This milling, however, was preferable for two reasons. First, MBM is heterogeneous at a fairly large scale; without reducing the particle size and mixing, it is unlikely that any small sample of MBM would be representative of the whole. Secondly, small particle size was a requirement for injecting the particles during the bioassay. The resulting fine powder was mixed well and split into ∼10 g samples by repeated cone-and-quartering. Samples were finally dried in a desiccator for 48 h to get rid of most of the humidity that interferes with the biodiesel reaction (in situ transesterification).
As a model for a rendered TSE-infected animal carcass we spiked 10 g MBM with 0.5 g hamster brain, froze it with liquid nitrogen, and homogenized the mixture using a mortar and pestle. Alkaline methanolysis was performed by shaking the MBM/ brain mixture vigorously in 24 ml 0.25 M sodium methoxide at 35°C for 2 hours. The study sample was spiked with brain from a scrapie infected hamster (263K), and an identical sample not subjected to the reaction served as a positive control. In parallel we performed a negative control reaction using MBM spiked with brain from a normal/uninfected hamster.
This alternate method of biodiesel production does not require prior lipid extraction but has been proven to convert lipids into Fatty acid methyl esters with high efficiency
The in situ transesterification of lipids to FAME typically leads to 3 different reaction phases or products which are easy to separate. The solid MBM residue was air dried overnight and crushed with mortar and pestle (without liquid nitrogen) to obtain a homogeneous sample. The solid MBM residue was suspended in 320 mM sucrose to 25 µg total protein/50 µl with gentle heating for 20 min at 40°C. The resulting non-homogenous suspension was passed through a 20 gauge needle, and then a 27 gauge needle, and some solid material was lost in each passage. The biodiesel and glycerol phases were simply diluted into 320 mM sucrose to an estimated amount of 25 µg total protein/50 µl (10-3 ID50). Under anesthesia, weanling female Syrian hamsters received 50 µl of intracranial inoculum. Inocula for toxicity studies were prepared as described above but reaction phases were diluted into 320 mM Sucrose at two different concentrations (25 µg and 0.25 µg total protein/50 µl) and only non infected MBM was used.
10% Brain homogenate was prepared at room temperature by homogenizing 1 g hamster brain with a Polytron homogenizer in 10 ml MES extraction buffer (60 mM n-Octyl-glucoside, 1% Triton-X 100, 25 mM MES-HCl, pH 6.5, 150 mM NaCl; all chemicals from Sigma-Aldrich Corp., St. Louis, Missouri, United States) containing one tablet of Roche complete mini protease inhibitor (Roche Corp., Basel, Switzerland) and 1 mM phenyl-methane-sulfonyl-fluoride (Sigma-Aldrich Corp.). The homogenate was precleared at 3000 rpm for 5 min in a centrifuge (Eppendorf Corp., Hamburg, Germany). The solution was diluted 1∶10 in the above described MES buffer and aliquoted for further use. For Western Blotting aliquots were loaded on a SDS-PAGE gel at 25 µg total protein/ lane. Protein content was measured by BCA using an Eppendorf Biophotometer.
Crude reaction fractions of biodiesel and glycerol were diluted with 4x sample loading buffer (SLB, Invitrogen Corp., Carlsbad, California, United States), heated to 70°C for 10 min and directly loaded onto a precast 4–12% gradient gel (Invitrogen Corp.). The non reacted brain spiked MBM and solid MBM residue each were boiled for 10 min in sample loading buffer and supernatants were taken off and analyzed by immunoblotting. Previous prepared inocula of the respective fractions (see above) were simply diluted and subjected to Western Blot analysis.
For detection of PrPsc infectious samples were digested with proteinase K solution (Roche Corp., 15 µg/µl final concentration) for 1 hour at 60°C.
Electrophoresis was performed using an XCell SureLock Mini-Cell (Invitrogen Corp.). Protein transfer was performed wet with a Mini Trans-blot Cell (Bio-Rad, Hercules, California, United States). PrP was detected using either monoclonal mAb 3F4 or monoclonal mAb IPC1 (Sigma-Aldrich Corp.). Protein bands were visualized with an anti-mouse HRP secondary antibody (Pierce Biotechnology Inc., Rockford, Illinois, United States) and enhanced chemiluminescent detection (Pierce Biotechnology Inc.).
Hamster brains were fixed in 10% neutral buffered formalin for 48 h then sunk in 30% sucrose. Coronal cryosections (5 µm) were serially collected, attached to glass slides, counterstained with Meyer's hematoxylin and Congo Red. Sections were evaluated on an inverted Leica DMI4000B microscope and digital images collected using an attached Leica DFC320 camera.
Cryo-sections were prepared from animals sacrificed 150 days post-inoculation, when the infected animal displayed severe scrapie symptoms.
Acute toxicity studies. To evaluate the acute toxicity and tolerable dose of the products of the biodiesel process, we performed Western blot and intracranial inoculation in hamsters using the reaction products from uninfected brain, after dilution of the 3 product phases (25 µg and 2.5 µg protein in 50 µl respectively). Western blot analysis (upper phase (Biodiesel c) lanes 2 and 3; interphase (Glycerol c), lanes 4 and 5; and lower phase (Solid MBM Residue c), lanes 6 and 7) failed to detect PrP in any inoculum. MBM spiked with 5% brain (MBM c, lanes 8-10, 25 µg, 2.5 µg and 0.25 µg in 50 µl respectively) and uninfected/normal brain homogenate (1% NBH, lane 1) served as controls. Coomassie stain (data not shown) showed a smear of protein present in all fractions except the upper phase (Biodiesel c), indicating the presence of proteinaceous material.
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