Subcritical Water Hydrolysis Effectively Reduces the In Vitro Seeding Activity of PrPSc but Fails to Inactivate the Infectivity of Bovine Spongiform Encephalopathy Prions.

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.

Ultrasensitive detection of PrP(Sc) in the cerebrospinal fluid and blood of macaques infected with bovine spongiform encephalopathy prion.

Prion diseases are characterized by the prominent accumulation of the misfolded form of a normal cellular protein (PrP(Sc)) in the central nervous system. The pathological features and biochemical properties of PrP(Sc) in macaque monkeys infected with the bovine spongiform encephalopathy (BSE) prion have been found to be similar to those of human subjects with variant Creutzfeldt-Jakob disease (vCJD). Non-human primate models are thus ideally suited for performing valid diagnostic tests and determining the efficacy of potential therapeutic agents. In the current study, we developed a highly efficient method for in vitro amplification of cynomolgus macaque BSE PrP(Sc). This method involves amplifying PrP(Sc) by protein misfolding cyclic amplification (PMCA) using mouse brain homogenate as a PrP(C) substrate in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrP(Sc) contained in the cerebrospinal fluid (CSF) and white blood cells (WBCs), as well as in the peripheral tissues of macaques that have been intracerebrally inoculated with the BSE prion. After clinical signs of the disease appeared in three macaques, we detected PrP(Sc) in the CSF by serial PMCA, and the CSF levels of PrP(Sc) tended to increase with disease progression. In addition, PrP(Sc) was detectable in WBCs at the clinical phases of the disease in two of the three macaques. Thus, our highly sensitive, novel method may be useful for furthering the understanding of the tissue distribution of PrP(Sc) in non-human primate models of CJD.

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.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Sensitive detection of scrapie prion protein in soil.

Prion diseases are fatal neurodegenerative disorders that are caused by infectious agents known as prions. Prions are composed primarily of the pathogenic prion protein isoform, PrP(Sc). Because significant levels of infectivity have been detected in excrement from animals infected with scrapie and chronic wasting disease, studies on the dynamics of PrP(Sc) levels in contaminated soil are needed to assess the possible horizontal transmission of prion diseases. Using protein misfolding cyclic amplification, we developed a sensitive detection method for scrapie PrP(Sc) that is mixed with soil. Our detection method has the advantage of not requiring extraction of PrP(Sc) from soil and could provide a sensitivity 1000 to 10,000 times higher than that obtained with an extraction-based method. In addition, we found that PrP(Sc) levels in experimentally contaminated agricultural soils declined to different extents over the course of a 6-month incubation period. Our method appears to be a very useful technique for monitoring PrP(Sc) levels in soil.