Rapid assessment of bovine spongiform encephalopathy prion inactivation by heat treatment in yellow grease produced in the industrial manufacturing process of meat and bone meals
© Yoshioka et al.; licensee BioMed Central Ltd. 2013
Received: 25 March 2013
Accepted: 3 July 2013
Published: 9 July 2013
Prions, infectious agents associated with transmissible spongiform encephalopathy, are primarily composed of the misfolded and pathogenic form (PrPSc) of the host-encoded prion protein. Because PrPSc retains infectivity after undergoing routine sterilizing processes, the cause of bovine spongiform encephalopathy (BSE) outbreaks are suspected to be feeding cattle meat and bone meals (MBMs) contaminated with the prion. To assess the validity of prion inactivation by heat treatment in yellow grease, which is produced in the industrial manufacturing process of MBMs, we pooled, homogenized, and heat treated the spinal cords of BSE-infected cows under various experimental conditions.
Prion inactivation was analyzed quantitatively in terms of the infectivity and PrPSc of the treated samples. Following treatment at 140°C for 1 h, infectivity was reduced to 1/35 of that of the untreated samples. Treatment at 180°C for 3 h was required to reduce infectivity. However, PrPSc was detected in all heat-treated samples by using the protein misfolding cyclic amplification (PMCA) technique, which amplifies PrPScin vitro. Quantitative analysis of the inactivation efficiency of BSE PrPSc was possible with the introduction of the PMCA50, which is the dilution ratio of 10% homogenate needed to yield 50% positivity for PrPSc in amplified samples.
Log PMCA50 exhibited a strong linear correlation with the transmission rate in the bioassay; infectivity was no longer detected when the log PMCA50 of the inoculated sample was reduced to 1.75. The quantitative PMCA assay may be useful for safety evaluation for recycling and effective utilization of MBMs as an organic resource.
KeywordsPrion inactivation Bovine spongiform encephalopathy Meat and bone meal Yellow grease Infectivity Protein misfolding cyclic amplification
Transmissible spongiform encephalopathies (TSEs), including scrapie in sheep and goats, chronic wasting disease (CWD) in deer and elk, bovine spongiform encephalopathy (BSE) in cattle, and Creutzfeldt–Jakob disease (CJD) in humans, are infectious and fatal neurodegenerative diseases . Proteinaceous infectious agents called prions are thought to be responsible for TSEs, which are characterized by the accumulation of the pathogenic form of prion protein (PrPSc) in the nervous tissues of infected subjects [2, 3]. PrPSc is a conformational isoform of the normal cellular prion protein (PrPC), which is rich in beta-sheet structures, insoluble in mild detergents, and resistant to protease digestion [4, 5].
Because prions retain infectivity after undergoing routine sterilization processes , contaminated meat and bone meals (MBMs) are suspected to be the source of BSE infection [7, 8]. MBMs are manufactured through a multi-step process involving the crushing of carcasses in a pre-breaker, heating at 120°C–140°C in yellow grease (lower-quality grades of tallow) in a cooker, and degreasing from solid material by an oil separator. To determine BSE prion inactivation during the manufacturing process of MBMs, industrial processes were replicated on a pilot scale by using BSE-infected brains, and the infectivity of processed materials in each step was investigated in detail . However, in reality, processing conditions for MBMs differ among rendering houses producing commercial MBMs. Since the efficiency of prion inactivation could be influenced by various factors such as treatment temperature, time, steam pressure in the cooker, size, water and fat contents of carcasses [10–13], it is difficult to identify the risks attributable to specific processing conditions. Furthermore, PrPSc retained in the manufacturing process of MBMs remains to be elucidated.
The governments of many countries prohibited the feeding of bovine MBMs following the feeding ban on MBMs in the United Kingdom. A prion detection method with high sensitivity and high accuracy must be developed so that MBMs can be used safely in the future. In addition, BSE prion is more resistant to physical and chemical treatments than are scrapie and CJD prions . Therefore, experiments using BSE-infected materials are essential for the assessment of BSE prion inactivation as they can be considered a worst case among prions. In recent years, it has become possible to perform in vitro amplification of PrPSc derived from various animals [15–21] by using protein misfolding cyclic amplification (PMCA) . We developed an ultrasensitive method for BSE PrPSc detection using potassium dextran sulfate (DSP) . The PMCA technique can also be used to quantitatively assess scrapie PrPSc[23–25], and our PMCA method can be applied as an effective test for the assessment of prion inactivation by monitoring residual BSE PrPSc. In the present study, we investigated efficiency of BSE prion inactivation following heat treatment in yellow grease by bioassay and quantitative PMCA.
Infectivity of heat-treated homogenates
Mean incubation time of TgBoPrP mice following intracerebral inoculation of titrated bovine spongiform encephalopathy (BSE)-infected spinal cord homogenate
10% Homogenate dilution
Transmission rate (diseased/total)
Mean incubation time ± SD (days)
242 ± 14
279 ± 12
322 ± 42
367 ± 53
736, 736, >790
Effects of various heat treatments in yellow grease on BSE-infected spinal cord homogenates
Transmission rate (diseased/total)
Mean incubation time ± SD (days)
Log reduction of infectivity
Log reduction of PMCA50
304 ± 13*,‡
382 ± 64, >790
471 ± 80*,†, >790
380 ± 25†,‡
PrPScdetection by PMCA
Quantitative analysis of PrPSc
Figure 1b shows the results of the amplification of each diluted sample of untreated BSE-infected spinal cord homogenate. PrPSc present in 10–7 dilution of the infected homogenate was detected in all tubes after three rounds of amplification. PrPSc signals were detected in three of the 10–8 and one of the 10–9 dilutions after three rounds of amplification. However, no additional tubes became positive for PrPSc in these dilutions after four rounds of amplification. No signals were detected in the more extreme dilution ranges even after four rounds of amplification. Thus, the PMCA50 of the 10% homogenate was calculated to be 108.5 units on the basis of the results obtained from the fourth round of amplification.
In this study, BSE prion inactivation was analyzed quantitatively in terms of infectivity and the PrPSc contents of the samples after heat treatment in yellow grease. Following treatment at 140°C for 1 h, which is the heat treatment condition generally used for carcasses in rendering houses in Japan, the infectivity of the BSE-infected spinal cord homogenate was reduced to at most 1/35 of that of the untreated control samples; furthermore, PrPSc retained its capability for in vitro propagation. Because carcasses are usually heat treated in closed cookers, prions are affected by the steam pressure from the water contained in the carcass. If a sufficient amount of water is present in the carcass, prion inactivation may proceed more efficiently in the cooker than under atmospheric pressure. However, some degree of BSE infectivity was still detected after autoclaving at 133°C in spiked raw materials with high infectivity levels . Furthermore, the precise effects of high-pressure steam on carcasses submerged in yellow grease are not known. Therefore, high-risk materials such as brains and spinal cord should be excluded from the rendering process for effective inactivation of BSE prion.
We previously examined residual infectivity and PrPSc after heat treatment of scrapie-infected hamster brains under various experimental conditions . The PMCA results were concordant with bioassay results. However, BSE PrPSc was detected in the samples treated at 180°C for 3 h, although infectivity was not detected in the bioassay. There are several possible explanations for this discrepancy between infectivity and PrPSc occurrence. For example, BSE PrPSc might contain various forms of PrPSc with different amplification properties and infectivity, and a PMCA-compatible form of PrPSc with low or no infectivity might predominate after heat treatment and be maintained over other forms throughout the amplification process. However, in the present study, the log PMCA50 values were strongly correlated with the transmission rate in the bioassay (Figure 3), suggesting that such PMCA-compatible but less-infectious PrPSc was not selectively amplified in vitro.
In our previous paper, we demonstrated our amplification system was highly sensitive and accurate, and no spontaneous generation of PrPSc was observed in the amplification of various kind of samples derived from uninfected animals . Determination of PMCA50 based on quadruplicate amplification was also done in our previous study , and we confirmed that similar PMCA50 values (around 1011 per gram) were obtained in two independent studies. In the present study, the PMCA50 of the BSE-infected spinal cords was estimated to be 1011.6 per gram, which is approximately 80,000-fold greater than the corresponding intracerebral LD50 per gram (106.7) determined by the bioassay. The PMCA50/LD50 per gram of BSE prion was considerably higher than those of scrapie prion strains (160–4000 fold) . If this ratio reflects the number of PrPSc particles that compose an infectious unit of prions, more PrPSc particles might participate in an infectious unit of BSE prions; moreover, such a large mass of PrPSc particles might be processed into several smaller ones with lower infectivity in vivo.
Another aspect of heat treatment in yellow grease is that higher-temperature treatments do not necessarily inactivate BSE prion more effectively. In the case of heat treatment for 1 h, the results of both the bioassay and PMCA indicate that BSE prion inactivation proceeded more effectively with treatment at 160°C rather than at 180°C. Samples treated at 180°C were dark brown, suggesting that the surface was scorched during treatment. In such high-temperature conditions, thermal conduction may be inhibited by scorching of the sample periphery, consequently requiring longer treatment time to reach thermal equilibrium in the sample. Extension of the treatment time to 3 h was actually necessary for the loss of infectivity. However, further studies are needed to confirm the above possibility.
In this study, we demonstrated that heat treatment at 180°C for 3 h is required for the loss of infectivity of BSE prion in grease heating in our experimental conditions. Furthermore, BSE PrPSc retains amplification ability even after such a treatment. The inactivation efficiency of BSE PrPSc could be quantitatively analyzed with the introduction of the PMCA50, which is strongly correlated with the transmission rate in the bioassay. The serial PMCA technique is more practical and less time consuming than bioassays, and may be applicable for monitoring residual PrPSc in the other steps of the manufacturing of MBMs and useful for safety evaluation for recycling and effective utilization of MBMs as an organic resource.
Experimental heat treatment procedure
All animal experiments were approved by the Animal Care and Use Committee of the National Institute of Animal Health (approval IDs: 450 and 08-008) in accordance with the Guidelines for Animal Transmissible Spongiform Encephalopathy Experiments of the Ministry of Agriculture, Forestry, and Fisheries of Japan. Spinal cords were obtained from four cows experimentally inoculated with BSE at the terminal stage of the disease. The infected materials were pooled and homogenized using a blender. Pure homogenate (0.5 g) was placed on a strip of aluminum foil (2 cm × 2 cm) and stored at –80°C until further use. For use, the homogenate with the aluminum foil was thawed at room temperature and then immersed in 15 mL yellow grease preheated to 140°C, 160°C, or 180°C in a ceramic crucible by using an electric heating device (ND-M11, Nissin Rika, Tokyo, Japan). The yellow grease used was obtained from a rendering house in Japan. The crucible was covered, and a thermosensor was inserted through a hole in the cover to monitor the temperature of the yellow grease. The yellow grease was, then, kept for 1 or 3 h at the desired temperature. The homogenate sample firmly adhered to the surface of aluminum foil and was not broken into pieces during the heat treatment. After the treatment, the homogenate with the aluminum foil was removed from the yellow grease with tweezers and placed on a paper towel for absorption of the excess yellow grease. The weights of the homogenates were reduced to 60–70% of their original weights. The resultant materials were thoroughly crushed with a mortar, and suspended in PBS at 10% (w/v). Insoluble materials were separated by brief centrifugation, and aqueous fraction was stored at –80°C until further use.
Infectivity titer using transgenic mice overexpressing bovine PrPC is generally 100-1000 times higher than that using cows. Therefore, more accurate estimation of BSE infectivity is able to be conducted by using such mice. The heat-treated samples were injected intracerebrally into six Tg(BoPrP)4092HOZ/Prnp0/0 (TgBoPrP) transgenic mice (20 μL per mouse) overexpressing bovine PrPC. To determine the infectivity titer, serial 10-fold dilutions of the 10% homogenate of the untreated spinal cords were prepared in PBS and injected intracerebrally into five to seven TgBoPrP mice (20 μL per mouse). After inoculation, the mice were evaluated daily for signs of infection. The lethal dose (LD50) was determined according to the 50% endpoint calculation method. Mean incubation times of the diseased mice were analyzed by one-way ANOVA and Tukey’s multiple comparison test.
Bovine PrPSc was amplified as described previously . Briefly, the brains of TgBoPrP transgenic mice and PrP knockout (PrP0/0) mice were homogenized separately in PBS containing 1% Triton X-100 and 4 m mol L–1 EDTA. After centrifugation at 4500 × g for 5 min, the supernatants were mixed in PrP0/0/TgBoPrP (5:1). A mixture containing 0.5% DSP was used as the PrPC substrate for PMCA.
The 10% homogenates of heat-treated samples were mixed at 1:9 with the PrPC substrate (total volume, 100 μL) in electron beam-irradiated polystyrene tubes. Amplification was performed in duplicate with a fully automatic cross-ultrasonic protein-activating apparatus (Elestein 070-CPR, Elekon Science, Chiba, Japan), which has a capacity to generate high ultrasonic power (700 W). PMCA amplification was performed by 40 cycles of sonication (3-s pulse oscillations repeated 5 times at 1-s intervals), followed by incubation at 37°C for 1 h with agitation. For serial PMCA, 1:5 dilution of the PMCA product and subsequent amplification was repeated twice.
To evaluate the inactivation efficiency of BSE PrPSc by heat treatment, the PMCA50, which is the dilution ratio of the 10% homogenate needed to yield 50% PrPSc positivity for amplified samples, was determined. Serial 10-fold dilutions of the 10% homogenate of the heat-treated and untreated samples were prepared and mixed 1:9 with the PrPC substrate (total volume, 80 μL) and amplified in electron beam-irradiated eight-strip polystyrene tubes (076-96, Elekon Science). Amplification was performed in quadruplicate using 40 cycles of sonication (pulse oscillation for 5 s, repeated 5 times at 1-s intervals), followed by incubation at 37°C for 1 h with agitation. For serial PMCA, 1:5 dilution of the amplified product and subsequent amplification was repeated 3 times. The PMCA50 was estimated from the results of the fourth round of amplification by using the 50% endpoint calculation method.
The amplified samples (10 μL) were mixed with 10 μL proteinase K solution (100 μg mL–1) and incubated at 37°C for 1 h. The digested samples were mixed with 20 μL 2× SDS sample buffer and incubated at 100°C for 5 min. The samples were separated by SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (Millipore, Bedford, MA). After the membrane was blocked, it was incubated for 30 min with a horseradish peroxidase (HRP)-conjugated T2 monoclonal antibody . After washing, the blotted membrane was developed using the Luminata Forte Western HRP Substrate (Millipore) according to the manufacturer’s instructions. Chemiluminescence signals were analyzed with a Light Capture System (Atto, Tokyo Japan).
The left hemispheres of the brains were fixed in 10% buffered formalin for neuropathological analysis. Coronal brain sections were immersed in 98% formic acid to reduce infectivity and embedded in paraffin wax. Sections (4 μm thick) were cut and stained with hematoxylin and eosin, and analyzed immunehistochemically as described previously .
Pathogenic form of prion protein
Bovine spongiform encephalopathy
Meat and bone meals
Protein misfolding cyclic amplification
Transmissible spongiform encephalopathies
Chronic wasting disease
Normal cellular prion protein.
We wish to thank the animal caretakers of the Prion Disease Research Center of the National Institute of Animal Health for their assistance. This study was funded by a grant from the Bovine Spongiform Encephalopathy Control Project of the Ministry of Agriculture, Forestry, and Fisheries of Japan.
- Collinge J: Prion diseases of humans and animals: their causes and molecular basis. Annu Rev Neurosci. 2001, 24: 519-550. 10.1146/annurev.neuro.24.1.519.PubMedView ArticleGoogle Scholar
- Prusiner SB: Molecular biology of prion disease. Science. 1991, 252 (5012): 1515-1522. 10.1126/science.1675487.PubMedView ArticleGoogle Scholar
- Prusiner SB: Prions. Proc Natl Acad Sci USA. 1998, 95 (23): 13363-13383. 10.1073/pnas.95.23.13363.PubMedPubMed CentralView ArticleGoogle Scholar
- Caughey BW, Dong A, Bhat KS, Ernst D, Hayes SF, Caughey WS: Secondary structure analysis of the scrapie-associated protein PrP 27-30 in water by infrared spectroscopy. Biochemistry. 1991, 30 (31): 7672-7680. 10.1021/bi00245a003.PubMedView ArticleGoogle Scholar
- Pan KM, Baldwin M, Nguyen J, Gasset M, Serban A, Groth D, Mehlhorn I, Huang Z, Fletterick RJ, Cohen FE, et al: Conversion of α-helics into β-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci USA. 1993, 90 (23): 10962-10966. 10.1073/pnas.90.23.10962.PubMedPubMed CentralView ArticleGoogle Scholar
- Taylor DM: Resistance of transmissible spongiform encephalopathy agents to decontamination. In Prions, A Challenge for Science, Medicine and Public Health System. Edited by Rabenau HF, Ciantl J, Doerr HW. Basel: Karger; 2001:58–67Google Scholar
- Wilesmith JW, Wells GA, Cranwell MP, Ryan JB: Bovine spongiform encephalopathy: epidemiological studies. Vet Rec. 1988, 123 (25): 638-644.PubMedGoogle Scholar
- Butler D: Statistics suggest BSE now ‘Europe-wide’. Nature. 1996, 382 (6586): 4-PubMedView ArticleGoogle Scholar
- Taylor DM: Inactivation of the bovine spongiform encephalopathy agent by rendering procedures. Vet Rec. 1995, 137 (24): 605-610.PubMedGoogle Scholar
- Taylor DM: Inactivation of prions by physical and chemical means. J Hosp Infect. 1999, 43 (Suppl 1): S69-S76.PubMedView ArticleGoogle Scholar
- Taylor DM, Fernie K, McConnell I, Steele PJ: Observations on thermostable subpopulations of the unconventional agents that cause transmissible degenerative encephalopathies. Vet Microbiol. 1998, 64 (1): 33-38. 10.1016/S0378-1135(98)00257-0.PubMedView ArticleGoogle Scholar
- Schreuder BEK, Geertsma RE, van Keulen LJ, van Asten JA, Enthoven P, Oberthür RC, de Koeijer AA, Osterhaus AD: Studies on the efficacy of hyperbaric rendering procedures in inactivation bovine spongiform encephalopathy (BSE) and scrapie agents. Vet Rec. 1998, 142 (18): 474-480. 10.1136/vr.142.18.474.PubMedView ArticleGoogle Scholar
- Muller H, Stitz L, Wille H, Prusiner SB, Riesner D: Influence of water, fat, glycerol on the mechanism of thermal prion inactivation. J Biol Chem. 2007, 282 (49): 35855-35867. 10.1074/jbc.M706883200.PubMedView ArticleGoogle Scholar
- Giles K, Glidden DV, Beckwith R, Seoanes R, Peretz D, DeArmond SJ, Prusiner SB: Resistance of bovine spongiform encephalopathy (BSE) prions to inactivation. PLoS Pathog. 2008, 4 (11): e1000206-10.1371/journal.ppat.1000206.PubMedPubMed CentralView ArticleGoogle Scholar
- Saá P, Castilla J, Soto C: Ultra-efficient replication of infectious prions by automated protein misfolding cyclic amplification. J Biol Chem. 2006, 281 (46): 35245-35252. 10.1074/jbc.M603964200.PubMedView ArticleGoogle Scholar
- Murayama Y, Yoshioka M, Yokoyama T, Iwamaru Y, Imamura M, Masujin K, Yoshiba S, Mohri S: Efficient in vitro amplification of a mouse-adapted scrapie prion protein. Neurosci Lett. 2007, 413 (3): 270-273. 10.1016/j.neulet.2006.11.056.PubMedView ArticleGoogle Scholar
- Kurt TD, Perrott MR, Wilusz CJ, Wilusz J, Supattapone S, Telling GC, Zabel MD, Hoover EA: Efficient in vitro amplification of chronic wasting disease PrPRES. J Virol. 2007, 81 (17): 9605-9608. 10.1128/JVI.00635-07.PubMedPubMed CentralView ArticleGoogle Scholar
- Thorne L, Terry LA: In vitro amplification of PrPSc derived from the brain and blood of sheep infected with scrapie. J Gen Virol. 2008, 89 (12): 3177-3184. 10.1099/vir.0.2008/004226-0.PubMedView ArticleGoogle Scholar
- Jones M, Peden AH, Prowse CV, Gröner A, Manson JC, Turner ML, Ironside JW, MacGregor IR, Head MW: In vitro amplification and detection of variant Creutzfeldt-Jakob disease PrPSc. J Pathol. 2007, 213 (1): 21-26. 10.1002/path.2204.PubMedView ArticleGoogle Scholar
- Murayama Y, Yoshioka M, Masujin K, Okada H, Iwamaru Y, Imamura M, Matsuura Y, Fukuda S, Onoe S, Yokoyama T, et al: Sulfated dextrans enhance in vitro amplification of bovine spongiform encephalopathy PrPSc and enable ultrasensitive detection of bovine PrPSc. PLoS One. 2010, 5 (10): e13152-10.1371/journal.pone.0013152.PubMedPubMed CentralView ArticleGoogle Scholar
- Yokoyama T, Takeuchi A, Yamamoto M, Kitamoto T, Ironside JW, Morita M: Heparin enhances the cell-protein misfolding cyclic amplification efficiency of variant Creutzfeldt-Jakob disease. Neurosci Lett. 2011, 498 (2): 119-123. 10.1016/j.neulet.2011.04.072.PubMedView ArticleGoogle Scholar
- Saborio GP, Permanne B, Soto C: Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature. 2001, 411 (6839): 810-813. 10.1038/35081095.PubMedView ArticleGoogle Scholar
- Chen B, Morales R, Barria MA, Soto C: Estimating prion concentration in fluids and tissues by quantitative PMCA. Nat Meth. 2010, 7 (7): 519-520. 10.1038/nmeth.1465.View ArticleGoogle Scholar
- Pritzkow S, Wagenführ K, Daus ML, Boerner S, Lemmer K, Thomzig A, Mielke M, Beekes M: Quantitative detection and biological propagation of scrapie seeding activity in vitro facilitate use of prions as model pathogens for disinfection. PLoS One. 2011, 6 (5): e20384-10.1371/journal.pone.0020384.PubMedPubMed CentralView ArticleGoogle Scholar
- Makarava N, Savtchenko R, Alexeeva I, Rohwer RG, Baskakov IV: Fast and ultrasensitive method for quantitating prion infectivity titre. Nat Commun. 2012, 3: 741-PubMedPubMed CentralView ArticleGoogle Scholar
- Matsuura Y, Ishikawa Y, Bo X, Murayama Y, Yokoyama T, Somerville RA, Kitamoto T, Mohri S: Quantitative analysis of wet-heat inactivation in bovine spongiform encephalopathy. Biochem Biophys Res Commun. 2013, 432 (1): 86-91. 10.1016/j.bbrc.2013.01.081.PubMedView ArticleGoogle Scholar
- Murayama Y, Yoshioka M, Horii H, Takata M, Yokoyama T, Sudo T, Sato K, Shinagawa M, Mohri S: Protein misfolding cyclic amplification as a rapid test for assessment of prion inactivation. Biochem Biophys Res Commun. 2006, 348 (2): 758-762. 10.1016/j.bbrc.2006.07.130.PubMedView ArticleGoogle Scholar
- Race R, Chesebro B: Scrapie infectivity found in resistant species. Nature. 1998, 392 (6678): 770-10.1038/33834.PubMedView ArticleGoogle Scholar
- Hill AF, Joiner S, Linehan J, Desbruslais M, Lantos PL, Collinge J: Species-barrier-independent prion replication in apparently resistant species. Proc Natl Acad Sci USA. 2000, 97 (18): 10248-10253. 10.1073/pnas.97.18.10248.PubMedPubMed CentralView ArticleGoogle Scholar
- Race R, Raines A, Raymond GJ, Caughey B, Chesebro B: Long-term subclinical carrier state precedes scrapie replication and adaptation in a resistant species: analogies to bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease in humans. J Virol. 2001, 75 (21): 10106-10112. 10.1128/JVI.75.21.10106-10112.2001.PubMedPubMed CentralView ArticleGoogle Scholar
- Scott MR, Safar J, Telling G, Nguyen O, Groth D, Torchia M, Koehler R, Tremblay P, Walther D, Cohen FE, et al: Identification of a prion protein epitope modulating transmission of bovine spongiform encephalopathy prions to transgenic mice. Proc Natl Acad Sci USA. 1997, 94 (26): 14279-14284. 10.1073/pnas.94.26.14279.PubMedPubMed CentralView ArticleGoogle Scholar
- Hayashi HK, Yokoyama T, Takata M, Iwamaru Y, Imamura M, Ushiki YK, Shinagawa M: The N-terminal cleavage site of PrPSc from BSE differs from that of PrPSc from scrapie. Biochem Biophys Res Commun. 2005, 328 (4): 1024-1027. 10.1016/j.bbrc.2005.01.065.PubMedView ArticleGoogle Scholar
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