Abstract

The global outbreak of bovine spongiform encephalopathy (BSE) has been attributed to the recycling of contaminated meat and bone meals (MBMs) as feed supplements. The use of MBMs has been prohibited in many countries; however, the development of a method for inactivating BSE prions could enable the efficient and safe use of these products as an organic resource. Subcritical water (SCW), which is water heated under pressure to maintain a liquid state at temperatures below the critical temperature (374°C), exhibits strong hydrolytic activity against organic compounds. In this study, we examined the residual in vitro seeding activity of protease-resistant prion protein (PrPSc) and the infectivity of BSE prions after SCW treatments. Spinal cord homogenates prepared from BSE-infected cows were treated with SCW at 230–280°C for 5–7.5 min and used to intracerebrally inoculate transgenic mice overexpressing bovine prion protein. Serial protein misfolding cyclic amplification (sPMCA) analysis detected no PrPSc in the SCW-treated homogenates, and the mice treated with these samples survived for more than 700 days without any signs of disease. However, sPMCA analyses detected PrPSc accumulation in the brains of all inoculated mice. Furthermore, secondary passage mice, which inoculated with brain homogenates derived from a western blotting (WB)-positive primary passage mouse, died after an average of 240 days, similar to mice inoculated with untreated BSE-infected spinal cord homogenates. The PrPSc accumulation and vacuolation typically observed in the brains of BSE-infected mice were confirmed in these secondary passage mice, suggesting that the BSE prions maintained their infectivity after SCW treatment. One late-onset case, as well as asymptomatic but sPMCA-positive cases, were also recognized in secondary passage mice inoculated with brain homogenates from WB-negative but sPMCA-positive primary passage mice. These results indicated that SCW-mediated hydrolysis was insufficient to eliminate the infectivity of BSE prions under the conditions tested.

Highlights

  • It is widely believed that protease-resistant, misfolded forms of prion proteins (PrPSc) comprise the infectious agents that cause prion diseases such as Creutzfeldt-Jacob disease (CJD) in humans, scrapie in sheep and goats, chronic wasting disease (CWD) in deer and elk, and bovine spongiform encephalopathy (BSE) in cattle [1]

  • No PrPSc signal was detected by conventional western blotting (WB) analysis in any of the subcritical water (SCW)-treated samples (Fig 1A), indicating that such treatments were sufficient to alter or break down the T2 epitope of the prion protein

  • We demonstrated that PrPSc derived from BSE-infected cattle could be efficiently amplified by Serial protein misfolding cyclic amplification (sPMCA) in the presence of DSP [31]

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Summary

Introduction

It is widely believed that protease-resistant, misfolded forms of prion proteins (PrPSc) comprise the infectious agents that cause prion diseases such as Creutzfeldt-Jacob disease (CJD) in humans, scrapie in sheep and goats, chronic wasting disease (CWD) in deer and elk, and bovine spongiform encephalopathy (BSE) in cattle [1]. Because contaminated MBMs are suspected to be the source of BSE infection, the governments of many countries have prohibited the feeding of ruminant MBMs. BSE prions are more resistant to inactivation treatments than scrapie and CJD prions [16]. The development of an effective method for enabling the utilization of bovine MBMs as an organic resource would require thorough evaluation of both the levels of PrPSc and the residual infectivity of BSE prions

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