Catherine Graham

Prion Strain Mutation Determined by Prion Protein Conformational Compatibility and Primary Structure

CWD Strain Variation So-called prion diseases are fatal neurogenerative disorders that include chronic wasting disease (CWD) found in deer and other cervids. Prion diseases are thought to be caused by infectious proteins (prions) in the absence of associated infectious DNA. Nevertheless, prion strains have been isolated that can mutate in the absence of nucleic acids, and these strain properties control the ability of prions to cross species barriers. Angers et al. (p. 1154, published online 13 May; see the Perspective by Collinge) address the issue of strain variation in the context of CWD. Whereas the host range of this contagious disease continues to expand, the prevalence of CWD strains has not been determined. Understanding CWD strain variation may be important in predicting and preventing any future risks to human health. The stability of two related strains is influenced by a species-specific amino acid difference in deer and elk prions. Prions are infectious proteins composed of the abnormal disease-causing isoform PrPSc, which induces conformational conversion of the host-encoded normal cellular prion protein PrPC to additional PrPSc. The mechanism underlying prion strain mutation in the absence of nucleic acids remains unresolved. Additionally, the frequency of strains causing chronic wasting disease (CWD), a burgeoning prion epidemic of cervids, is unknown. Using susceptible transgenic mice, we identified two prevalent CWD strains with divergent biological properties but composed of PrPSc with indistinguishable biochemical characteristics. Although CWD transmissions indicated stable, independent strain propagation by elk PrPC, strain coexistence in the brains of deer and transgenic mice demonstrated unstable strain propagation by deer PrPC. The primary structures of deer and elk prion proteins differ at residue 226, which, in concert with PrPSc conformational compatibility, determines prion strain mutation in these cervids.

Cell-Based Quantification of Chronic Wasting Disease Prions

ABSTRACT Cell-based measurement of prion infectivity is currently restricted to experimental strains of mouse-adapted scrapie. Having isolated cell cultures with susceptibility to prions from diseased elk, we describe a modification of the scrapie cell assay allowing evaluation of prions causing chronic wasting disease, a naturally occurring transmissible spongiform encephalopathy. We compare this cervid prion cell assay to bioassays in transgenic mice, the only other existing method for quantification, and show this assay to be a relatively economical and expedient alternative that will likely facilitate studies of this important prion disease.

The identification of disease-induced biomarkers in the urine of BSE infected cattle

BackgroundThe bovine spongiform encephalopathy (BSE) epidemic and the emergence of a new human variant of Creutzfeldt-Jakob Disease (vCJD) have led to profound changes in the production and trade of agricultural goods. The rapid tests currently approved for BSE monitoring in slaughtered cattle are all based on the detection of the disease related isoform of the prion protein, PrPd, in brain tissue and consequently are only suitable for post-mortem diagnosis. Objectives: In instances such as assessing the health of breeding stock for export purposes where post-mortem testing is not an option, there is a demand for an ante-mortem test based on a matrix or body fluid that would permit easy access and repeated sampling. Urine and urine based analyses would meet these requirements.ResultsTwo dimensional differential gel eletrophoresis (2D-DIGE) and mass spectrometry analyses were used to identify proteins exhibiting differential abundance in the urine of BSE infected cattle and age matched controls over the course of the disease. Multivariate analyses of protein expression data identified a single protein able to discriminate, with 100% accuracy, control from infected samples. In addition, a subset of proteins were able to predict with 85% ± 13.2 accuracy the time post infection that the samples were collected.ConclusionThese results suggest that in principle it is possible to identify biomarkers in urine useful in the diagnosis, prognosis and monitoring of disease progression of transmissible spongiform encephalopathy diseases (TSEs).

Biodegradation of Prions in Compost

Composting may serve as a practical and economical means of disposing of specified risk materials (SRM) or animal mortalities potentially infected with prion diseases (transmissible spongiform encephalopathies, TSE). Our study investigated the degradation of prions associated with scrapie (PrP(263K)), chronic waste disease (PrP(CWD)), and bovine spongiform encephalopathy (PrP(BSE)) in lab-scale composters and PrP(263K) in field-scale compost piles. Western blotting (WB) indicated that PrP(263K), PrP(CWD), and PrP(BSE) were reduced by at least 2 log10, 1-2 log10, and 1 log10 after 28 days of lab-scale composting, respectively. Further analysis using protein misfolding cyclic amplification (PMCA) confirmed a reduction of 2 log10 in PrP(263K) and 3 log10 in PrP(CWD). Enrichment for proteolytic microorganisms through the addition of feather keratin to compost enhanced degradation of PrP(263K) and PrP(CWD). For field-scale composting, stainless steel beads coated with PrP(263K) were exposed to compost conditions and removed periodically for bioassays in Syrian hamsters. After 230 days of composting, only one in five hamsters succumbed to TSE disease, suggesting at least a 4.8 log10 reduction in PrP(263K) infectivity. Our findings show that composting reduces PrP(TSE), resulting in one 50% infectious dose (ID50) remaining in every 5600 kg of final compost for land application. With these considerations, composting may be a viable method for SRM disposal.

Biodegradation of specified risk material and fate of scrapie prions in compost

Composting may be a viable alternative to rendering and land filling for the disposal of specified risk material (SRM) provided that infectious prion proteins (PrPTSE) are inactivated. This study investigated the degradation of SRM and the fate of scrapie prions (PrPSc) over 28 days in laboratory-scale composters, with and without feathers in the compost matrices. Compost was mixed at day 14 to generate a second heating cycle, with temperatures exceeding 65°C in the first cycle and 50°C in the second cycle. Approximately 63% and 77% of SRM was degraded after the first and second cycles, respectively. Inclusion of feathers in the compost matrices did not alter compost properties during composting other than increasing (P < 0.05) total nitrogen and reducing (P < 0.05) the C/N ratio. However, addition of feathers enhanced (P < 0.05) SRM degradation by 10% upon completion of experiment. Scrapie brain homogenates were spiked into manure at the start of composting and extracted using sodium dodecyl sulphate followed by detection using Western blotting (WB). Prior to composting, PrPSc was detectable in manure with 1–2 log10 sensitivity, but was not observable after 14 or 28 days of composting. This may have been due to either biological degradation of PrPSc or the formation of complexes with compost components that precluded its detection.

Detergents modify proteinase K resistance of PrPSc in different transmissible spongiform encephalopathies (TSEs)

Prion diseases are diagnosed by the detection of their proteinase K-resistant prion protein fragment (PrP(Sc)). Various biochemical protocols use different detergents for the tissue preparation. We found that the resistance of PrP(Sc) against proteinase K may vary strongly with the detergent used. In our study, we investigated the influence of the most commonly used detergents on eight different TSE agents derived from different species and distinct prion disease forms. For a high throughput we used a membrane adsorption assay to detect small amounts of prion aggregates, as well as Western blotting. Tissue lysates were prepared using DOC, SLS, SDS or Triton X-100 in different concentrations and these were digested with various amounts of proteinase K. Detergents are able to enhance or diminish the detectability of PrP(Sc) after proteinase K digestion. Depending on the kind of detergent, its concentration - but also on the host species that developed the TSE and the disease form or prion type - the detectability of PrP(Sc) can be very different. The results obtained here may be helpful during the development or improvement of a PrP(Sc) detection method and they point towards a detergent effect that can be additionally used for decontamination purposes. A plausible explanation for the detergent effects described in this article could be an interaction with the lipids associated with PrP(Sc) that may stabilize the aggregates.

Factors affecting the accuracy of urine-based biomarkers of BSE

BackgroundTransmissible spongiform encephalopathy diseases are untreatable, uniformly fatal degenerative syndromes of the central nervous system that can be transmitted both within as well as between species. The bovine spongiform encephalopathy (BSE) epidemic and the emergence of a new human variant of Creutzfeldt-Jakob disease (vCJD), have profoundly influenced beef production processes as well as blood donation and surgical procedures. Simple, robust and cost effective diagnostic screening and surveillance tools are needed for both the preclinical and clinical stages of TSE disease in order to minimize both the economic costs and zoonotic risk of BSE and to further reduce the risk of secondary vCJD.ObjectiveUrine is well suited as the matrix for an ante-mortem test for TSE diseases because it would permit non-invasive and repeated sampling. In this study urine samples collected from BSE infected and age matched control cattle were screened for the presence of individual proteins that exhibited disease specific changes in abundance in response to BSE infection that might form the basis of such an ante-mortem test.ResultsTwo-dimensional differential gel electrophoresis (2D-DIGE) was used to identify proteins exhibiting differential abundance in two sets of cattle. The known set consisted of BSE infected steers and age matched controls throughout the course of the disease. The blinded unknown set was composed of BSE infected and control samples of both genders, a wide range of ages and two different breeds. Multivariate analyses of individual protein abundance data generated classifiers comprised of the proteins best able to discriminate between the samples based on disease state, breed, age and gender.ConclusionDespite the presence of confounding factors, the disease specific changes in abundance exhibited by a panel of urine proteins permitted the creation of classifiers able to discriminate between control and infected cattle with a high degree of accuracy.

Disease-specific motifs can be identified in circulating nucleic acids from live elk and cattle infected with transmissible spongiform encephalopathies

To gain insight into the disease progression of transmissible spongiform encephalopathies (TSE), we searched for disease-specific patterns in circulating nucleic acids (CNA) in elk and cattle. In a 25-month time-course experiment, CNAs were isolated from blood samples of 24 elk (Cervus elaphus) orally challenged with chronic wasting disease (CWD) infectious material. In a separate experiment, blood-sample CNAs from 29 experimental cattle (Bos taurus) 40 months post-inoculation with clinical bovine spongiform encephalopathy (BSE) were analyzed according to the same protocol. Next-generation sequencing provided broad elucidation of sample CNAs: we detected infection-specific sequences as early as 11 months in elk (i.e. at least 3 months before the appearance of the first clinical signs) and we established CNA patterns related to BSE in cattle at least 4 months prior to clinical signs. In elk, a progression of CNA sequence patterns was found to precede and correlate with macro-observable disease progression, including delayed CWD progression in elk with PrP genotype LM. Some of the patterns identified contain transcription-factor-binding sites linked to endogenous retroviral integration. These patterns suggest that retroviruses may be connected to the manifestation of TSEs. Our results may become useful for the early diagnosis of TSE in live elk and cattle.

Urine Proteins Identified by Two-Dimensional Differential Gel Electrophoresis Facilitate the Differential Diagnoses of Scrapie

The difficulty in developing a diagnostic assay for Creutzfeldt - Jakob disease (CJD) and other transmissible spongiform encephalopathies (TSEs) stems in part from the fact that the infectious agent is an aberrantly folded form of an endogenous cellular protein. This precludes the use of the powerful gene based technologies currently applied to the direct detection of other infectious agents. To circumvent this problem our research objective has been to identify a set of proteins exhibiting characteristic differential abundance in response to TSE infection. The objective of the present study was to assess the disease specificity of differentially abundant urine proteins able to identify scrapie infected mice. Two-dimensional differential gel electrophoresis was used to analyze longitudinal collections of urine samples from both prion-infected mice and a transgenic mouse model of Alzheimers disease. The introduction of fluorescent dyes, that allow multiple samples to be co-resolved and visualized on one two dimensional gel, have increased the accuracy of this methodology for the discovery of robust protein biomarkers for disease. The accuracy of a small panel of differentially abundant proteins to correctly classify an independent naïve sample set was determined. The results demonstrated that at the time of clinical presentation the differential abundance of urine proteins were capable of identifying the prion infected mice with 87% sensitivity and 93% specificity. The identity of the diagnostic differentially abundant proteins was investigated by mass spectrometry.

Can plants serve as a vector for prions causing chronic wasting disease

Prions, the causative agent of chronic wasting disease (CWD) enter the environment through shedding of bodily fluids and carcass decay, posing a disease risk as a result of their environmental persistence. Plants have the ability to take up large organic particles, including whole proteins, and microbes. This study used wheat (Triticum aestivum L.) to investigate the uptake of infectious CWD prions into roots and their transport into aerial tissues. The roots of intact wheat plants were exposed to infectious prions (PrPTSE) for 24 h in three replicate studies with PrPTSE in protein extracts being detected by western blot, IDEXX and Bio-Rad diagnostic tests. Recombinant prion protein (PrPC) bound to roots, but was not detected in the stem or leaves. Protease-digested CWD prions (PrPTSE) in elk brain homogenate interacted with root tissue, but were not detected in the stem. This suggests wheat was unable to transport sufficient PrPTSE from the roots to the stem to be detectable by the methods employed. Undigested PrPTSE did not associate with roots. The present study suggests that if prions are transported from the roots to the stems it is at levels that are below those that are detectable by western blot, IDEXX or Bio-Rad diagnostic kits.