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 CreutzfeldtJakob 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.
BackgroundPrions, 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.ResultsPrion 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.ConclusionsLog 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.
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