Bovine Spongiform Encephalopathy PrP Research Articles

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.

Bovine spongiform encephalopathy and food safety

Bovine spongiformencephalopathy (BSE) is a fatal diseaseof cattle, caused by infective proteins known as prions. A prion (PrP is a mis-folded isoform of the glycoprotein PrP, which is highly expressed in the nervous system. Prions replicate by coercing PrP to refold into PrP. The BSE epidemic was propagated by rendering dead cattle to producemealwhichwas then included in cattle feed. Consumption of BSE PrP from contaminated beef resulted in over 200 human cases of variant Creutzfeldt-Jakob (vCJD) disease, which is invariably fatal. There were rare cases of person-to-person vCJD transmission by blood transfusion. Variant CJD is now very rare, due to adoption of measures that prevent the feeding of ruminant protein to ruminants and the contamination of beef with the tissues that harbour PrP. Beef from countries with these control systems are safe for human consumption.

Risk of Prion Disease Transmission through Bovine‐Derived Bone Substitutes: A Systematic Review

Despite the causal association between variant Creutzfeldt - Jakob disease and bovine spongiform encephalopathy (BSE), bovine origin graft materials are widely used during dental surgical procedures. The aim of this study was to assess the risk of BSE transmission through anorganic bovine bone substitutes. Electronic database of MEDLINE was searched to identify relevant studies regarding our focused questions, presence of BSE prion infectivity in raw bovine bone, BSE prion inactivation by bone substitute manufacturing process, protein contents in anorganic bovine bone substitutes, and validity of current BSE diagnostic methods. Search terms yielded 1,704 titles. After title/abstract screening and duplicates removal, 36 full-text articles were screened for inclusion. A total of 16 studies were included in the final analysis. No eligible studies were identified regarding the efficacy of BSE prion inactivation by the treatments used for anorganic bovine bone manufacturing. BSE infectivity and PrP(Sc) , pathological prion, were detected in bovine bone marrow and serum samples. Proteins were detected in Tutoplast® (bovine), Bio-Oss®, and tibia samples treated at the similar condition for Bio-Oss deproteinization. Inconsistent results of different BSE diagnostic tests were not unusual findings (Iwata et al. 2006; Arnold et al. 2007; Murayama et al. 2010), and a study by Balkema-Buschmann and colleagues showed an apparent discrepancy between BSE infectivity and detection of PrP(27-30), the current surrogate marker for prion disease infectivity. This review indicates that bovine-derived graft biomaterials may carry a risk of prion transmission to patients.

Bovine macrophage degradation of scrapie and BSE PrP Sc

Transmissible spongiform encephalopathies (TSEs), such as bovine spongiform encephalopathy (BSE) and scrapie, display long incubation periods before PrP Sc accumulates in the central neuronal system (CNS). The precise role that phagocytic cells, such as macrophages, play in prion pathogenesis is uncertain. In this study, the involvement of bovine macrophages at the early stage of prion infection was studied. Brain homogenates of mouse scrapie and BSE were degraded sequentially in the bovine macrophage cell line, Bo120, and freshly prepared in monocyte-derived macrophages from peripheral blood. Mouse scrapie brain homogenates degraded in Bo120 cells were inoculated intraperitoneally to C57BL mice, showing that the degree of cellular degradation (2 h, 10, 28, and 36 d) correlated with survival periods (288, 303, 324, and 340 d, respectively). Partial colocalizations of PrP and lysosomes were observed in Bo120 cells by confocal microscopy. These results suggest that bovine macrophages have the ability to take up and degrade PrP Sc, resulting in decreased TSE infectivity in mice.

Immunohistochemical characteristics of disease-associated PrP are not altered by host genotype or route of inoculation following infection of sheep with bovine spongiform encephalopathy

It has previously been reported that disease-associated prion protein (PrP(d)) derived from natural scrapie and from sheep infected experimentally with bovine spongiform encephalopathy (BSE) differed in respect of their immunohistochemical and immunoblotting properties. For BSE, however, these initial observations were restricted to orally challenged sheep of the ARQ/ARQ PrP genotype. Here, extended examinations were performed on 28 sheep that developed neurological signs after BSE experimental infection by one of three routes. Intracerebrally infected ARQ/ARQ sheep showed more widespread and abundant accumulations of PrP(d) in tissues of the lymphoreticular system (LRS) than VRQ/VRQ animals, whereas no peripheral PrP(d) was detected in ARR/ARR sheep. The intensity and dissemination of PrP(d) accumulation in LRS tissues were less than those found previously in orally dosed sheep. AHQ/AHQ sheep challenged orally and ARQ/AHQ and ARQ/ARQ animals infected intravenously showed similar LRS-tissue PrP(d) distributions and levels to those of ARQ/ARQ sheep infected intracerebrally. The patterns of intra- and extracellular immunoreactivity to different PrP antibodies in brain and LRS tissues and the immunoblotting characteristics of PrP(res) from brain samples remained constant, irrespective of the route of inoculation and the PrP genotype, and were the same as described previously for ARQ/ARQ sheep dosed orally with BSE. These results suggest that the intracellular truncation of BSE PrP(d) and the proteinase K cleavage site of BSE PrP(res) are not altered by PrP genotype or by route of inoculation and that, therefore, screening tests based on these properties can be applied to identify potential sheep BSE cases occurring naturally.

The N-terminal cleavage site of PrP Sc from BSE differs from that of PrP Sc from scrapie

Heterogeneity in transmissible spongiform encephalopathy is thought to have derived from conformational variation in an abnormal isoform of the prion protein (PrP Sc). To characterize PrP Sc in bovine spongiform encephalopathy (BSE) and scrapie, we analyzed the newly generated N-terminus of PrP Sc isoforms by digestion with proteinase K (PK). With a lower concentration of PK, the terminal amino acid of BSE PrP Sc converged at N 96. Under the same conditions, however, the terminal amino acid of scrapie PrP Sc was G 81 or G 85. Furthermore, with an increase of PK concentration, the N-terminal amino acid was shifted and converged at G 89. The results suggest that the PK cleavage site of BSE PrP Sc is uniform and is different from the cleavage site of scrapie PrP Sc.

Molecular analysis of the abnormal prion protein during coinfection of mice by bovine spongiform encephalopathy and a scrapie agent.

Molecular features of the proteinase K-resistant prion protein (PrP res) may discriminate among prion strains, and a specific signature could be found during infection by the infectious agent causing bovine spongiform encephalopathy (BSE). To investigate the molecular basis of BSE adaptation and selection, we established a model of coinfection of mice by both BSE and a sheep scrapie strain (C506M3). We now show that the PrP res features in these mice, characterized by glycoform ratios and electrophoretic mobilities, may be undistinguishable from those found in mice infected with scrapie only, including when mice were inoculated by both strains at the same time and by the same intracerebral inoculation route. Western blot analysis using different antibodies against sequences near the putative N-terminal end of PrP res also demonstrated differences in the main proteinase K cleavage sites between mice showing either the BSE or scrapie PrP res profile. These results, which may be linked to higher levels of PrP res associated with infection by scrapie, were similar following a challenge by a higher dose of the BSE agent during coinfection by both strains intracerebrally. Whereas PrP res extraction methods used allowed us to distinguish type 1 and type 2 PrP res, differing, like BSE and scrapie, by their electrophoretic mobilities, in the same brain region of some patients with Creutzfeldt-Jakob disease, analysis of in vitro mixtures of BSE and scrapie brain homogenates did not allow us to distinguish BSE and scrapie PrP res. These results suggest that the BSE agent, the origin of which remains unknown so far but which may have arisen from a sheep scrapie agent, may be hidden by a scrapie strain during attempts to identify it by molecular studies and following transmission of the disease in mice.