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ABSTRACT: BACKGROUND: Prions, the causative agents of the transmissible spongiform encephalopathies, are notoriously difficult to inactivate. Current decontamination recommendations by the World Health Organization include prolonged exposure to 1 N sodium hydroxide or > 20,000 ppm sodium hypochlorite, or autoclaving. For decontamination of large stainless steel surfaces and equipment as in abattoirs, for example, these methods are harsh or unsuitable. The current study was designed to evaluate the effectiveness of a commercial product containing sodium percarbonate to inactivate prions. Samples of mouse brain infected with a mouse-adapted strain of the scrapie agent (RML) were exposed to a sodium percarbonate-based product (SPC-P). Treated samples were evaluated for abnormal prion protein (PrPSc)-immunoreactivity by western blot analysis, and residual infectivity by mouse bioassay. RESULTS: Exposure to a 21% solution of SPC-P or a solution containing either 2.1% or 21% SPC-P in combination with sodium dodecyl sulfate (SDS) resulted in increased proteinase K sensitivity of PrPSc. Limited reductions in infectivity were observed depending on treatment condition. A marginal effect on infectivity was observed with SPC-P alone, but an approximate 2--3 log10 reduction was observed with the addition of SDS, though exposure to SDS alone resulted in an approximate 2 log10 reduction. CONCLUSIONS: This study demonstrates that exposure of a mouse-adapted scrapie strain to SPC-P does not eliminate infectivity, but does render PrPSc protease sensitive.
BMC Veterinary Research 01/2013; 9(1):8. · 2.00 Impact Factor
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ABSTRACT: Cattle could be exposed to the agent of chronic wasting disease (CWD) through contact with infected farmed or free-ranging cervids or exposure to contaminated premises. The purpose of the current study was to assess the potential for CWD derived from elk to transmit to cattle after intracranial inoculation. Calves (n = 14) were inoculated with brain homogenate derived from elk with CWD to determine the potential for transmission and to define the clinicopathologic features of disease. Cattle were necropsied if clinical signs occurred or at the end of the study (49 months postinoculation; MPI). Clinical signs of poor appetite, weight loss, circling, and bruxism occurred in 2 cattle (14%) at 16 and 17 MPI, respectively. Accumulation of abnormal prion protein (PrP(Sc)) occurred in only the 2 clinically affected cattle and was confined to the central nervous system, with the most prominent immunoreactivity in midbrain, brainstem, and hippocampus with lesser immunoreactivity in the cervical spinal cord. The rate of transmission was lower than in cattle inoculated with CWD derived from mule deer (38%) or white-tailed deer (86%). Additional studies are required to fully assess the potential for cattle to develop CWD through a more natural route of exposure, but a low rate of transmission after intracranial inoculation suggests that risk of transmission through other routes is low. A critical finding is that if CWD did transmit to exposed cattle, currently used diagnostic techniques would detect and differentiate it from other prion diseases in cattle based on absence of spongiform change, distinct pattern of PrP(Sc) deposition, and unique molecular profile.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 09/2012; · 1.21 Impact Factor
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ABSTRACT: Transmissible spongiform encephalopathies (TSEs), including scrapie in sheep (Ovis aries), are fatal neurodegenerative diseases caused by the misfolding of the cellular prion protein (PrP C) into a â-rich conformer (PrP Sc) that accumulates into higher-order structures in the brain and other tissues. Distinct strains of TSEs exist, characterized by different pathologic profiles upon passage into rodents and representing distinct conformations of PrP Sc . One biochemical method of distinguishing strains is the stability of PrP Sc as determined by unfolding in guanidine hydrochloride (GdnHCl), which is tightly and positively correlated with the incubation time of disease upon passage into mice. Here, we utilize a rapid, protease-free version of the stability assay to characterize naturally occurring scrapie samples, including a fast-acting scrapie inoculum for which incubation time is highly dependent on the amino acid at codon 136 of the prion protein. We utilize the stability methodology to identify the presence of two distinct isolates in the inoculum, and compare isolate properties to those of a host-stabilized reference scrapie isolate (NADC 13-7) in order to assess the stability/incubation time correlation in a natural host system. We demonstrate the utility of the stability methodology in characterizing TSE isolates throughout serial passage in livestock, which is applicable to a range of natural host systems, including strains of bovine spongiform encephalopathy and chronic wasting disease. This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
PLoS ONE 08/2012; 7(8):e43060. · 4.09 Impact Factor
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ABSTRACT: Sheep scrapie susceptibility or resistance is a function of genotype, with polymorphisms at codon 171 in the sheep prion gene playing a major role. Glutamine (Q) at codon 171 contributes to scrapie susceptibility, while arginine (R) is associated with resistance. In some breeds, lysine (K) occurs at codon 171, but its effect on scrapie resistance has not been determined. Charge and structural similarities between K and R suggest that they may contribute to prion disease susceptibility in a similar way, but studies have not been performed to confirm this. The purpose of the current study was to compare susceptibility and incubation times of AA(136)RR(154)QQ(171) (where the letter denotes the amino acid and the number the position) with AA(136)RR(154)QK(171) sheep after inoculation with scrapie. Barbado AA(136)RR(154)QQ(171) and AA(136)RR(154)QK(171) sheep were inoculated with scrapie intracerebrally to assess their susceptibility to scrapie. After inoculation, sheep were observed daily for clinical signs and were euthanized and necropsied after clinical signs were unequivocal. Tissues were collected at necropsy for immunohistochemistry and Western blot analyses. The QQ(171) sheep had clinical signs approximately 12 months after inoculation, whereas QK(171) animals had an average incubation time of 30 months to onset of clinical signs. The distribution of abnormal prion protein was similar in QQ(171) and QK(171) sheep. Results of the study indicate that sheep with a single K allele at codon 171 are susceptible to scrapie but with a prolonged incubation time. Work is currently underway to examine relative scrapie susceptibility or resistance of KK(171) sheep.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 05/2012; 24(3):554-8. · 1.21 Impact Factor
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ABSTRACT: The majority of bovine spongiform encephalopathy (BSE) cases have been ascribed to the classical form of the disease. H-type and L-type BSE cases have atypical molecular profiles compared to classical BSE and are thought to arise spontaneously. However, one case of H-type BSE was associated with a heritable E211K mutation in the prion protein gene. The purpose of this study was to describe transmission of this unique isolate of H-type BSE when inoculated into a calf of the same genotype by the intracranial route. Electroretinograms were used to demonstrate preclinical deficits in retinal function, and optical coherence tomography was used to demonstrate an antemortem decrease in retinal thickness. The calf rapidly progressed to clinical disease (9.4 months) and was necropsied. Widespread distribution of abnormal prion protein was demonstrated within neural tissues by western blot and immunohistochemistry. While this isolate is categorized as BSE-H due to a higher molecular mass of the unglycosylated PrP(Sc) isoform, a strong labeling of all 3 PrP(Sc) bands with monoclonal antibodies 6H4 and P4, and a second unglycosylated band at approximately 14 kDa when developed with antibodies that bind in the C-terminal region, it is unique from other described cases of BSE-H because of an additional band 23 kDa demonstrated on western blots of the cerebellum. This work demonstrates that this isolate is transmissible, has a BSE-H phenotype when transmitted to cattle with the K211 polymorphism, and has molecular features that distinguish it from other cases of BSE-H described in the literature.
PLoS ONE 01/2012; 7(6):e38678. · 4.09 Impact Factor
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Eric M Nicholson
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ABSTRACT: Diagnosis of prion disease is primarily through immunodetection of the infectious agent. Typically, 2 distinct procedures are recommended for a definitive diagnosis, with immunohistochemistry and Western blot providing the most information as to the specific isolate in question. In the past, these approaches required formalin-fixed, paraffin-embedded tissue and fresh or frozen tissue, respectively; however, methods have been developed that allow for use of fixed tissue for Western blot. The present study describes a method of enriching PrP(Sc) in formalin-fixed, paraffin-embedded tissues prior to Western blot analysis for the detection of PrP(Sc). With this modified procedure, 5 times the previously reported sample size may be used for analysis, greatly enhancing the sensitivity of this procedure.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 07/2011; 23(4):790-2. · 1.21 Impact Factor
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ABSTRACT: Prion diseases or transmissible spongiform encephalopathies (TSEs) of animals include scrapie of sheep and goats; transmissible mink encephalopathy (TME); chronic wasting disease (CWD) of deer, elk and moose; and bovine spongiform encephalopathy (BSE) of cattle. The emergence of BSE and its spread to human beings in the form of variant Creutzfeldt-Jakob disease (vCJD) resulted in interest in susceptibility of cattle to CWD, TME and scrapie. Experimental cross-species transmission of TSE agents provides valuable information for potential host ranges of known TSEs. Some interspecies transmission studies have been conducted by inoculating disease-causing prions intracerebrally (IC) rather than orally; the latter is generally effective in intraspecies transmission studies and is considered a natural route by which animals acquire TSEs. The "species barrier" concept for TSEs resulted from unsuccessful interspecies oral transmission attempts. Oral inoculation of prions mimics the natural disease pathogenesis route whereas IC inoculation is rather artificial; however, it is very efficient since it requires smaller dosage of inoculum, and typically results in higher attack rates and reduces incubation time compared to oral transmission. A species resistant to a TSE by IC inoculation would have negligible potential for successful oral transmission. To date, results indicate that cattle are susceptible to IC inoculation of scrapie, TME, and CWD but it is only when inoculated with TME do they develop spongiform lesions or clinical disease similar to BSE. Importantly, cattle are resistant to oral transmission of scrapie or CWD; susceptibility of cattle to oral transmission of TME is not yet determined.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 05/2011; 23(3):407-20. · 1.21 Impact Factor
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ABSTRACT: Final observations on experimental transmission of chronic wasting disease (CWD) from elk (Cervus elaphus nelsoni) and white-tailed deer (Odocoileus virginianus) to fallow deer (Dama dama) are reported herein. During the 5-year study, 13 fawns were inoculated intracerebrally with CWD-infected brain material from white-tailed deer (n = 7; Group A) or elk (n = 6; Group B), and 3 other fawns were kept as uninoculated controls (Group C). As described previously, 3 CWD-inoculated deer were euthanized at 7.6 mo post-inoculation (MPI). None revealed presence of abnormal prion protein (PrP(d)) in their tissues. At 24 (Group A) and 26 (Group B) MPI, 2 deer were necropsied. Both animals had a small focal accumulation of PrP(d) in their midbrains. Between 29 and 37 MPI, 3 other deer (all from Group A) were euthanized. The 5 remaining deer became sick and were euthanized between 51 and 60 MPI (1 from Group A and 4 from Group B). Microscopic lesions of spongiform encephalopathy (SE) were observed in only these 5 animals; however, PrP(d) was detected in tissues of the central nervous system by immunohistochemistry, Western blot, and by commercial rapid test in all animals that survived beyond 24 MPI. This study demonstrates that intracerebrally inoculated fallow deer not only amplify CWD prions, but also develop lesions of spongiform encephalopathy.
Canadian journal of veterinary research = Revue canadienne de recherche vétérinaire 04/2011; 75(2):152-6. · 0.94 Impact Factor
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ABSTRACT: Formalin-fixed paraffin-embedded tissue is regularly employed in the diagnosis of transmissible spongiform encephalopathies (TSE) by immunohistochemistry (IHC), the standard by which all other TSE diagnostic protocols are judged. While IHC affords advantages over diagnostic approaches that typically utilize fresh or frozen tissue, such as Western blot and ELISA, the process of fixing, staining, and analyzing individual sections by hand does not allow for rapid or high throughput screening. However, preservation of tissues in formalin is not dependent upon the availability of refrigeration.
Formalin-fixed paraffin-embedded tissues from TSE transmission studies of scrapie in sheep, chronic wasting disease in white-tailed deer or transmissible mink encephalopathy in cattle were cut at 5 μm thickness. Samples containing the tissue equivalent of as little as one 5 μm section can be used to readily discriminate positive from negative samples.
This approach cannot replace IHC but may be used along with IHC as both a more rapid and readily high throughput screen where fresh or frozen tissues are not available or impractical.
BMC Research Notes 01/2011; 4:432.
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ABSTRACT: Prion diseases are fatal neurodegenerative diseases resulting from misfolding of normal cellular prion (PrP(C)) into an abnormal form of scrapie prion (PrP(Sc)). The cellular mechanisms underlying the misfolding of PrP(C) are not well understood. Since cellular prion proteins harbor divalent metal-binding sites in the N-terminal region, we examined the effect of manganese on PrP(C) processing in in vitro models of prion disease. Exposure to manganese significantly increased PrP(C) levels both in cytosolic and in membrane-rich fractions in a time-dependent manner. Manganese-induced PrP(C) upregulation was independent of messenger RNA transcription or stability. Additionally, manganese treatment did not alter the PrP(C) degradation by either proteasomal or lysosomal pathways. Interestingly, pulse-chase analysis showed that the PrP(C) turnover rate was significantly altered with manganese treatment, indicating increased stability of PrP(C) with the metal exposure. Limited proteolysis studies with proteinase-K further supported that manganese increases the stability of PrP(C). Incubation of mouse brain slice cultures with manganese also resulted in increased prion protein levels and higher intracellular manganese accumulation. Furthermore, exposure of manganese to an infectious prion cell model, mouse Rocky Mountain Laboratory-infected CAD5 cells, significantly increased prion protein levels. Collectively, our results demonstrate for the first time that divalent metal manganese can alter the stability of prion proteins and suggest that manganese-induced stabilization of prion protein may play a role in prion protein misfolding and prion disease pathogenesis.
Toxicological Sciences 02/2010; 115(2):535-46. · 4.65 Impact Factor
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ABSTRACT: Scrapie is a naturally occurring fatal neurodegenerative disease of adult sheep and goats, one of a group of mammalian diseases known as transmissible spongiform encephalopathies (TSE) or prion diseases. Immunoassays that identify disease-associated prion protein (PrP Sc) are integral to the diagnosis of scrapie and other prion diseases. Results obtained by either immunohistochemistry (IHC) or Western blot (WB) assay are generally adequate for the definitive diagnosis. Approved or accepted methods for WB diagnosis of TSEs requires the use of fresh or frozen nonfixed tissue samples, whereas formalin-fixed, paraffin-embedded tissue is required for the localization of PrP Sc by IHC. Because disparate processing methods are used for these accepted diagnostic techniques, separate tissue samples are collected from the same animal. Occasions arise in which there is either insufficient quantity of tissue available to complete analysis by both techniques or initial tissue processing is incompatible with one of the assays. Also, results between the assays may differ because of the vagaries of sampling, especially in case material that contains moderate-to-low levels of PrP Sc. The present article describes a method to conduct a WB assay from the same paraffin-embedded brainstem sample used for the IHC diagnosis of experimentally induced sheep scrapie.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 08/2008; 20(4):522-6. · 1.21 Impact Factor
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ABSTRACT: Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy (TSE) of cattle. Classical BSE is associated with ingestion of BSE-contaminated feedstuffs. H- and L-type BSE, collectively known as atypical BSE, differ from classical BSE by displaying a different disease phenotype and they have not been linked to the consumption of contaminated feed. Interestingly, the 2006 US H-type atypical BSE animal had a polymorphism at codon 211 of the bovine prion gene resulting in a glutamic acid to lysine substitution (E211K). This substitution is analogous a human polymorphism associated with the most prevalent form of heritable TSE in humans, and it is considered to have caused BSE in the 2006 US atypical BSE animal. In order to determine if this amino acid change is a heritable trait in cattle, we sequenced the prion alleles of the only known offspring of this animal, a 2-year-old heifer.
Sequence analysis revealed that both the 2006 US atypical BSE animal and its 2-year-old heifer were heterozygous at bovine prion gene nucleotides 631 through 633 for GAA (glutamic acid) and AAA (lysine). Both animals carry the E211K polymorphism, indicating that the allele is heritable and may persist within the cattle population.
This is the first evidence that the E211K polymorphism is a germline polymorphism, not a somatic mutation, suggesting BSE may be transmitted genetically in cattle. In the event that E211K proves to result in a genetic form of BSE, this would be the first indication that all 3 etiologic forms of TSEs (spontaneous, hereditary, and infectious) are present in a non-human species. Atypical BSE arising as both genetic and spontaneous disease, in the context of reports that at least some forms of atypical BSE can convert to classical BSE in mice, suggests a cattle origin for classical BSE.
PLoS ONE 02/2008; 3(8):e2912. · 4.09 Impact Factor
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ABSTRACT: Clinical signs of prion disease are not specific and include a variety of differential diagnoses. Serological tests and nucleic acid-based detection methods are not applicable to prion-disease-agent detection because of the unusual nature of the infectious agent. Prion-disease diagnosis is primarily conducted by means of immunodetection of the infectious agent, typically by at least 2 distinct procedures with immunohistochemistry and Western blot being the most informative. These approaches differ in the need for formalin-fixed and frozen or fresh tissue respectively. This work describes a method for the detection of the disease-associated isoform of the prion protein by Western blot using formalin-fixed tissues. The approach requires only minimal modification of existing Western-blot procedures and could readily be incorporated into existing detection schemes for confirmatory purposes when fresh or frozen tissues are unavailable.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 10/2007; 19(5):548-52. · 1.21 Impact Factor
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ABSTRACT: Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy of cattle, first detected in 1986 in the United Kingdom and subsequently in other countries. It is the most likely cause of variant Creutzfeldt-Jakob disease (vCJD) in humans, but the origin of BSE has not been elucidated so far. This report describes the identification and characterization of two cases of BSE diagnosed in the United States. Case 1 (December 2003) exhibited spongiform changes in the obex area of the brainstem and the presence of the abnormal form of the prion protein, PrP(Sc), in the same brain area, by immunohistochemistry (IHC) and Western blot analysis. Initial suspect diagnosis of BSE for case 2 (November 2004) was made by a rapid ELISA-based BSE test. Case 2 did not exhibit unambiguous spongiform changes in the obex area, but PrP(Sc) was detected by IHC and enrichment Western blot analysis in the obex. Using Western blot analysis, PrP(Sc) from case 1 showed molecular features similar to typical BSE isolates, whereas PrP(Sc) from case 2 revealed an unusual molecular PrP(Sc) pattern: molecular mass of the unglycosylated and monoglycosylated isoform was higher than that of typical BSE isolates and case 2 was strongly labeled with antibody P4, which is consistent with a higher molecular mass. Sequencing of the prion protein gene of both BSE-positive animals revealed that the sequences of both animals were within [corrected] the range of the prion protein gene sequence diversity previously reported for cattle.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 03/2007; 19(2):142-54. · 1.21 Impact Factor
