Eliminating transmissibility of bovine spongiform encephalopathy by dry-heat treatment.

Bovine spongiform encephalopathy (BSE) prion is more resistant to heat inactivation compared to other prions, but the effect of heat inactivation has been reported to differ depending on the BSE-contaminated tissue state or heating type. We aimed to evaluate the secure level of inactivation of original BSE transmissibility by dry-heating. Cattle tissues affected with BSE were subjected to dry-heat treatment for 20 min at various temperatures ranging from 150 to 1000 °C. To assess the inactivation effect, we conducted protein misfolding cyclic amplification (PMCA) and follicular dendritic cell (FDC) assays in transgenic mice expressing bovine prion protein genes. Under dry-heating at 600 °C or higher, BSE cattle tissues lost their transmissibility in transgenic mice. In contrast, transmissibility was detected in the cattle tissues treated at temperatures of 400 °C or lower through the FDC assay combined with PMCA. In this study, we confirmed that transmissibility was eliminated in BSE-affected cattle tissues by dry-heating at 600 °C or higher.

BSE infectivity survives burial for five years with only limited spread.

The carcasses of animals infected with bovine spongiform encephalopathy (BSE), scrapie or chronic wasting disease (CWD) that remain in the environment (exposed or buried) may continue to act as reservoirs of infectivity. We conducted two experiments under near-field conditions to investigate the survival and dissemination of BSE infectivity after burial in a clay or sandy soil. BSE infectivity was either contained within a bovine skull or buried as an uncontained bolus of BSE-infected brain. Throughout the five-year period of the experiment, BSE infectivity was recovered in similar amounts from heads exhumed annually from both types of soil. Very low levels of infectivity were detected in the soil immediately surrounding the heads, but not in samples remote from them. Similarly, there was no evidence of significant lateral movement of infectivity from the buried bolus over 4 years although there was a little vertical movement in both directions. However, bioassay analysis of limited numbers of samples of rain water that had drained through the bolus clay lysimeter indicated that infectivity was present in filtrates. sPMCA analysis also detected low levels of PrPSc in the filtrates up to 25 months following burial, raising the concern that leakage of infectivity into ground water could occur. We conclude that transmissible spongiform encephalopathy infectivity is likely to survive burial for long periods of time, but not to migrate far from the site of burial unless a vector or rain water drainage transports it. Risk assessments of contaminated sites should take these findings into account.

Quantitative analysis of wet-heat inactivation in bovine spongiform encephalopathy.

The bovine spongiform encephalopathy (BSE) agent is resistant to conventional microbial inactivation procedures and thus threatens the safety of cattle products and by-products. To obtain information necessary to assess BSE inactivation, we performed quantitative analysis of wet-heat inactivation of infectivity in BSE-infected cattle spinal cords. Using a highly sensitive bioassay, we found that infectivity in BSE cattle macerates fell with increase in temperatures from 133°C to 150°C and was not detected in the samples subjected to temperatures above 155°C. In dry cattle tissues, infectivity was detected even at 170°C. Thus, BSE infectivity reduces with increase in wet-heat temperatures but is less affected when tissues are dehydrated prior to the wet-heat treatment. The results of the quantitative protein misfolding cyclic amplification assay also demonstrated that the level of the protease-resistant prion protein fell below the bioassay detection limit by wet-heat at 155°C and higher and could help assess BSE inactivation. Our results show that BSE infectivity is strongly resistant to wet-heat inactivation and that it is necessary to pay attention to BSE decontamination in recycled cattle by-products.

Characterization of the effect of heat on agent strains of the transmissible spongiform encephalopathies.

The causal agents of the transmissible spongiform encephalopathy (TSE) diseases, sometimes called prion diseases, are characterized by high resistance to inactivation with heat. Results from thermal inactivation experiments on nine TSE strains, seven passaged in two PrP genotypes, showed differences in sensitivity to heat inactivation ranging over 17 °C. In addition, the rate of inactivation with increasing temperature varied between TSE models. In some cases passage in an alternative PrP genotype had little effect on the resulting inactivation properties, but for others the infectious agent was inactivated at lower temperatures. No strain with higher thermostability properties was selected. The effect of mixing two TSE strains, to see whether their properties were affected through interaction with each other, was also examined. The results showed that both strains behaved as expected from the behaviour of the unmixed controls, and that the strain responsible for inducing TSE disease could be identified. There was no evidence of a direct effect on intrinsic strain properties. Overall, the results illustrate the diversity in properties of TSE strains. They require intrinsic molecular properties of TSE agents to accommodate high resistance to inactivation and a mechanism, independent of the host, to directly encode these differences. These findings are more readily reconciled with models of TSE agents with two separate components, one of which is independent of the host and comprises a TSE-specific nucleic acid, than with models based solely on conformational changes to a host protein.

Transport of the pathogenic prion protein through soils.

Transmissible spongiform encephalopathies (TSEs) are progressive neurodegenerative diseases and include bovine spongiform encephalopathy of cattle, chronic wasting disease (CWD) of deer and elk, scrapie in sheep and goats, and Creutzfeldt-Jakob disease in humans. An abnormally folded form of the prion protein (designated PrP(TSE)) is typically associated with TSE infectivity and may constitute the major, if not sole, component of the infectious agent. Transmission of CWD and scrapie is mediated in part by an environmental reservoir of infectivity. Soil appears to be a plausible candidate for this reservoir. The transport of TSE agent through soil is expected to influence the accessibility of the pathogen to animals after deposition and must be understood to assess the risks associated with burial of infected carcasses. We report the results of saturated column experiments designed to evaluate PrP(TSE) transport through five soils with relatively high sand or silt contents and low organic carbon content. Protease-treated TSE-infected brain homogenate was used as a model for PrP(TSE) present in decomposing infected tissue. Synthetic rainwater was used as the eluent. All five soils retained PrP(TSE); no detectable PrP(TSE) was eluted over more than 40 pore volumes of flow. Lower bound apparent attachment coefficients were estimated for each soil. Our results suggest that TSE agent released from decomposing tissues to soils with low organic carbon content would remain near the site of initial deposition. In the case of infected carcasses deposited on the land surface, this may result in local sources of infectivity to other animals.

TSE agent strains and PrP: reconciling structure and function.

The molecular structures of the infectious agents that cause transmissible spongiform encephalopathy (TSE) diseases are still not known despite the current wide acceptance of the prion hypothesis as the basis for their resolution. Here, data supporting and challenging the prion hypothesis in relation to both the biochemical and biological properties of TSE infectious agents are discussed. The need for the independent transmission of TSE agent-specific genetic information is described and the requirements for the molecule to carry this information are proposed. Such a molecule is likely to be a small nucleic acid encoding information to determine the diversity of the pathogenesis of TSE agents.

Comparative studies on the thermostability of five strains of transmissible-spongiform-encephalopathy agent.

The causal infectious agents of TSEs (transmissible spongiform encephalopathies or prion diseases) are renowned for their resistance to complete inactivation. Survival of TSE infectivity after autoclaving potentially compromises many procedures where TSE infectivity may be present, including surgical instrument sterilization. In the present study, the heat inactivation properties of five different TSE agents were tested in a variety of experiments by exposing them to a range of heat inactivation conditions. Although TSE infectivity was reduced after heating to 200 degrees C in a hot air oven, substantial amounts of infectivity remained. Unlike wet heat inactivation, no TSE strain-dependent differences were observed in the reduction in the amounts of infectivity produced by dry heat inactivation. However, the incubation periods of mice infected with one dry heated TSE strain, ME7, were substantially prolonged, whereas there was little or no effect for two other TSE models. Varying autoclaving conditions for three TSE strains between 132 and 138 degrees C, and times of exposure between 30 and 120 min, had little or no effect on the recovery of TSE infectivity. The results illustrate the limitations of TSE agent inactivation using heat-based methods. The results support the hypothesis that the structures of TSE agents are stabilized during heat-inactivation procedures, rendering them much more refractory to inactivation. This may occur through dehydration of the causal agents, specifically through the removal of the water of solvation from agent structures and hence stabilize interactions between prion protein and TSE agent-specific ligands.

Microdissection: a method developed to investigate mechanisms involved in transmissible spongiform encephalopathy pathogenesis.

BACKGROUND: The transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases affecting both human and animals. The neuroanatomical changes which occur in the central nervous system (CNS) of TSE infected animals include vacuolation, gliosis, neuronal loss and the deposition of a disease specific protein, PrPSc. Experimental murine models of scrapie, a TSE of sheep, have revealed that pathology may be confined to specific brain areas with targeting of particular neuronal subsets depending on route of injection and scrapie isolate. To assess the biochemical changes which are taking place in these targeted areas it was necessary to develop a reliable sampling procedure (microdissection) which could be used for a variety of tests such as western blotting and magnetic resonance spectroscopy. METHODS: The method described is for the microdissection of murine brains. To assess the usefulness of this dissection technique for producing similar sample types for analysis by various down-stream biochemical techniques, the areas dissected were analysed for PrPSc by western blotting and compared to immunocytochemical (ICC) techniques. RESULTS: Results show that the method generates samples yielding a consistent protein content which can be analysed for PrPSc. The areas in which PrPSc is found by western blotting compares well with localisation visualised by immunocytochemistry. CONCLUSION: The microdisssection method described can be used to generate samples suitable for a range of biochemical techniques. Using these samples a range of assays can be carried out which will help to elucidate the molecular and cellular mechanisms underlying TSE pathogenesis. The method would also be useful for any study requiring the investigation of discrete areas within the murine brain.

How independent are TSE agents from their hosts?

Central to understanding the nature TSE agents (or prions) is how their genetic information is distinguished from the host. Are TSEs truly infectious diseases with host-independent genomes, or are they aberrations of a host component derived from the host genome? Recent experiments tested whether glycosylation of host PrP affects TSE strain characteristics. Wild-type mice were infected with 3 TSE strains passaged through transgenic mice with PrP devoid of glycans at 1 or both N-glycosylation sites. Strain-specific characteristics of 1 TSE strain changed but did not change for 2 others. Changes resulted from the selection of mutant TSE strains in a novel replicative environment. In general the properties of established TSEs support the genetic independence of TSE agents from the host, and specifically the primary structure of PrP does not directly encode TSE agent properties. However sporadic TSEs, challenge this independency. The prion hypothesis explains emerging TSEs relatively successfully but poorly accounts for the diversity and mutability of established TSE strains, or how many different infectious conformations are sustained thermodynamically. Research on early changes in RNA expression and events at the ribosome may inform the debate on TSE agent properties and their interaction with host cell machinery.

Fate of prions in soil: detergent extraction of PrP from soils.

The transmissible spongiform encephalopathies (TSEs) are caused by infectious agents whose structures have not been fully characterized but include abnormal forms of the host protein PrP, designated PrP(Sc), which are deposited in infected tissues. The transmission routes of scrapie and chronic wasting disease (CWD) seem to include environmental spread in their epidemiology, yet the fate of TSE agents in the environment is poorly understood. There are concerns that, for example, buried carcasses may remain a potential reservoir of infectivity for many years. Experimental determination of the environmental fate requires methods for assessing binding/elution of TSE infectivity, or its surrogate marker PrP(Sc), to and from materials with which it might interact. We report a method using Sarkosyl for the extraction of murine PrP(Sc), and its application to soils containing recombinant ovine PrP (recPrP). Elution properties suggest that PrP binds strongly to one or more soil components. Elution from a clay soil also required proteinase K digestion, suggesting that in the clay soil binding occurs via the N-terminal of PrP to a component that is absent from the sandy soils tested.

Transmissible spongiform encephalopathy strain, PrP genotype and brain region all affect the degree of glycosylation of PrPSc.

Transmissible spongiform encephalopathies (TSEs), sometimes known as prion diseases, are caused by an infectious agent whose molecular properties have not been determined. Traditionally, different strains of TSE diseases are characterized by a series of phenotypic properties after passage in experimental animals. More recently it has been recognized that diversity in the degree to which an abnormal form of the host protein PrP, denoted PrP(Sc), is glycosylated and the migration of aglycosyl forms of PrP(Sc) on immunoblots may have some differential diagnostic potential. It has been recognized that these factors are affected by the strain of TSE agent but also by other factors, e.g. location within the brain. This study shows in some cases, but not others, that host PrP genotype has a major influence on the degree of PrP(Sc) glycosylation and migration on gels and provides further evidence of the effect of brain location. Accordingly both the degree of glycosylation and the apparent molecular mass of PrP(Sc) may be of some value for differential diagnosis between TSE strains, but only when host effects are taken into account. Furthermore, the data inform the debate about how these differences arise, and favour hypotheses proposing that TSE agents affect glycosylation of PrP during its biosynthesis.

Inactivation of the bovine-spongiform-encephalopathy (BSE) agent by the acid and alkaline processes used in the manufacture of bone gelatine.

A validation study was carried out to determine the capacity of the traditional acid and alkaline processes used in the manufacture of bovine bone gelatine to remove and/or inactivate the transmissible agent that causes BSE (bovine spongiform encephalopathy). Using an accurately scaled down laboratory process that precisely mimicked the minimum conditions of the industrial processes, gelatine (gelatin) was manufactured from industrial starting material that had been spiked with mouse brain infected with the 301V strain of mouse-passaged BSE agent. Clearance factors were determined by titrating the infectivity levels of the infected mouse brain tissue, the gelatine extracts, and the final sterilized gelatine solution. The infectivity level of the spiked starting material was 10(8.4) mouse intracerebral ID(50)/kg (ID(50) is the dose at which half of the challenged animals were infected). Clearance factors of 10(2.6) and 10(3.7) ID(50) were demonstrated for the first stages of the acid and alkaline processes respectively during which the bones are converted to crude gelatine. It was further demonstrated that the complete acid and alkaline processes both reduced infectivity to undetectable levels, giving clearance factors of >/=10(4.8) ID(50) for the acid process, and >/=10(4.9) ID(50) for the alkaline process.

Thermostability of mouse-passaged BSE and scrapie is independent of host PrP genotype: implications for the nature of the causal agents.

Five experimentally maintained strains of scrapie and BSE agents have been passaged in two PrP genotypes of mice. Brain macerates were autoclaved at 126 degrees C and the levels of surviving infectivity were measured by titration. There was a large difference in the survival properties of transmissible spongiform encephalopathy (TSE) infectivity between TSE strains. PrP genotype had little effect. Phenotypic properties of the TSE strains were not affected with the exception that with one strain (ME7), incubation periods of the heated sample were longer than the controls given equivalent doses. It is concluded that PrP is probably not responsible for differences in thermostability between strains. More likely, a host-independent molecule which differs in covalent structure between strains accounts for these properties.

Characterization of thermodynamic diversity between transmissible spongiform encephalopathy agent strains and its theoretical implications.

Some transmissible spongiform encephalopathy (TSE) (or "prion") strains, notably those derived from bovine spongiform encephalopathy, are highly resistant to total inactivation by heat. When three TSE strains derived from sheep with scrapie were heated, little inactivation took place at low temperatures, but at higher temperatures, considerable inactivation occurred. The temperature at which substantial inactivation first occurred varied according to TSE strain, and it was calculated to be 70 degrees C for the 22C strain, 84 degrees C for ME7, and 97 degrees C for 22A by fitting the data to a model based on competition between a destructive and a protective reaction. However, PrP(Sc) from mice infected with a range of TSE strains retained similar resistance to proteinase K digestion after heating to below or above these temperatures, showing that the properties of PrP(Sc) responsible for proteinase resistance do not correlate with those conferring thermostability on the TSE agent. The simplest explanation of these data is that the causal agent contains a macromolecular component that is structurally independent of the host, that it varies covalently between TSE strains, and that it is protected by other macromolecular components. The model is in accord with the virino hypothesis, which proposes a host-independent informational molecule protected by the host protein PrP.