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Chronic wasting disease (CWD) is a fatal prion disease that can infect deer, elk and moose. CWD has now been detected in 26 states of the USA, 3 Canadian provinces, South Korea, Norway, Sweden and Finland. CWD continues to spread from endemic areas, and new foci of infections are frequently detected. As increasing numbers of cervids become infected, the likelihood for human exposure increases. To date, no cases of CWD infection in humans have been confirmed, but experience with the BSE zoonosis in the United Kingdom suggests exposure to CWD should be minimized. Specifically, hunters, meat processors and others in contact with tissues from potentially CWD-infected cervids need a practical method to decontaminate knives, saws and other equipment. Prions are notoriously difficult to inactivate, and most effective methods require chemicals or sterilization processes that are either dangerous, caustic, expensive or not readily available. Although corrosive, sodium hypochlorite (bleach) is widely available and affordable and has been shown to inactivate prion agents including those that cause scrapie, bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. In the current study, we confirm that bleach is an effective disinfectant for CWD prions and establish minimum times and bleach concentrations to eliminate prion seeding activity from stainless steel and infected brain homogenate solutions. We found that a five-minute treatment with a 40% dilution of household bleach was effective at inactivating CWD seeding activity from stainless-steel wires and CWD-infected brain homogenates. However, bleach was not able to inactivate CWD seeding activity from solid tissues in our studies. [ABSTRACT FROM AUTHOR]

Prions cause transmissible infectious diseases in humans and animals and have been found to be transmissible by blood transfusion even in the presymptomatic stage. However, the concentration of prions in body fluids such as blood and urine is extremely low; therefore, direct diagnostic tests on such specimens often yield false-negative results. Quantitative preanalytical prion enrichment may significantly improve the sensitivity of prion assays by concentrating trace amounts of prions from large volumes of body fluids. Here, we show that beads conjugated to positively charged peptoids not only captured PrP aggregates from plasma of prion-infected hamsters, but also adsorbed prion infectivity in both the symptomatic and preclinical stages of the disease. Bead absorbed prion infectivity efficiently transmitted disease to transgenic indicator mice. We found that the readout of the peptoid-based misfolded protein assay (MPA) correlates closely with prion infectivity in vivo, thereby validating the MPA as a simple, quantitative, and sensitive surrogate indicator of the presence of prions. The reliable and sensitive detection of prions in plasma will enable a wide variety of applications in basic prion research and diagnostics. [ABSTRACT FROM AUTHOR]

The diagnosis of chronic wasting disease (CWD) relies on demonstration of the disease-associated misfolded CWD prion protein (PrPCWD) in brain or retropharyngeal lymph node tissue by immunodetection methods, e.g. ELISA and immunohistochemistry (IHC). The success of these methods relies on a quality sample of tissues, which requires both anatomical knowledge and considerable dissection to collect. As the prevalence of CWD continues to increase globally, the development of fast and cost-effective methods to detect the disease is vital to facilitate CWD detection and surveillance. To address these issues, we have evaluated third eyelids from CWD-infected deer and elk using real-time quaking induced conversion (RT-QuIC). We identified prion seeding activity in third eyelids in 24 of 25 (96%) CWD-infected white-tailed deer (Odocoileus virginianus). We detected RT-QuIC positivity in the third eyelid as early as 1 month after experimental CWD exposure. In addition, we identified prion seeding activity in third eyelids of 18 of 25 (72%) naturally exposed asymptomatic CWD-positive rocky mountain elk (Cervus canadensis nelson). We compared CWD detection by RT-QuIC and IHC in third eyelid, retropharyngeal lymph node, and brain in 10 deer in early symptomatic stage of disease. IHC detected PrPCWD deposition in third eyelid lymphoid follicles in 5 of 10 deer (50%) whereas third eyelids of all 10 animals were positive by RT-QuIC. This difference reflected in part a lower requirement for lymphoid follicle presence for seeding activity detection by RT-QuIC. In conclusion, RT-QuIC analysis of the third eyelid, an easily accessed tissue, has potential to advance CWD detection and testing compliance. [ABSTRACT FROM AUTHOR]

Prion diseases are members of neurodegenerative protein misfolding diseases (NPMDs) that include Alzheimer’s, Parkinson’s and Huntington diseases, amyotrophic lateral sclerosis, tauopathies, traumatic brain injuries, and chronic traumatic encephalopathies. No known therapeutics extend survival or improve quality of life of humans afflicted with prion disease. We and others developed a new approach to NPMD therapy based on reducing the amount of the normal, host-encoded protein available as substrate for misfolding into pathologic forms, using RNA interference, a catabolic pathway that decreases levels of mRNA encoding a particular protein. We developed a therapeutic delivery system consisting of small interfering RNA (siRNA) complexed to liposomes and addressed to the central nervous system using a targeting peptide derived from rabies virus glycoprotein. These liposome-siRNA-peptide complexes (LSPCs) cross the blood-brain barrier and deliver PrP siRNA to neuronal cells to decrease expression of the normal cellular prion protein, PrPC, which acts as a substrate for prion replication. Here we show that LSPCs can extend survival and improve behavior of prion-infected mice that remain immunotolerant to treatment. LSPC treatment may be a viable therapy for prion and other NPMDs that can improve the quality of life of patients at terminal disease stages. [ABSTRACT FROM AUTHOR]

Prion protein (PrPC) is a protease-sensitive and soluble cell surface glycoprotein expressed in almost all mammalian cell types. PrPSc, a protease-resistant and insoluble form of PrPC, is the causative agent of prion diseases, fatal and transmissible neurogenerative diseases of mammals. Prion infection is initiated via either ingestion or inoculation of PrPSc or when host PrPC stochastically refolds into PrPSc. In either instance, the early events that occur during prion infection remain poorly understood. We have used transgenic mice expressing mouse PrPC tagged with a unique antibody epitope to monitor the response of host PrPC to prion inoculation. Following intracranial inoculation of either prion-infected or uninfected brain homogenate, we show that host PrPC can accumulate both intra-axonally and within the myelin membrane of axons suggesting that it may play a role in axonal loss following brain injury. Moreover, in response to the inoculation host PrPC exhibits an increased insolubility and protease resistance similar to that of PrPSc, even in the absence of infectious prions. Thus, our results raise the possibility that damage to the brain may be one trigger by which PrPC stochastically refolds into pathogenic PrPSc leading to productive prion infection. [ABSTRACT FROM AUTHOR]

Sporadic Creutzfeldt-Jakob disease (sCJD) is a transmissible, rapidly progressive and fatal neurodegenerative disease. The transmissible agent linked to sCJD is composed of the misfolded form of the host-encoded prion protein. The combination of histopathological and biochemical analyses has allowed the identification and sub-classification of six sCJD subtypes. This classification depends on the polymorphic variability of codon 129 of the prion protein gene and the PrPres isotype, and appears to be associated with neuropathological and clinical features. Currently, sCJD subtyping is only fully achievable post mortem. However, a rapid and non-invasive method for discriminating sCJD subtypes in vita would be invaluable for the clinical management of affected individuals, and for the selection of participants for clinical trials. The CSF analysis by Real Time Quaking Induced Conversion (RT-QuIC) reaction is the most sensitive and specific ante mortem sCJD diagnostic test available to date, and it is used by a number of laboratories internationally. RT-QuIC takes advantage of the natural replication mechanisms of prions by template-induced misfolding, employing recombinant prion protein as reaction substrate. We asked whether epitope mapping, of the RT-QuIC reaction products obtained from seeding RT-QuIC with brain and CSF samples from each of the six molecular subtypes of sCJD could be employed to distinguish them and therefore achieve in vita sCJD molecular subtyping. We found that it is possible to distinguish the RT-QuIC products generated by sCJD biological samples from the ones generated by spontaneous conversion in the negative controls, but that different sCJD subtypes generate very similar, if not identical RT-QuIC reaction products. We concluded that whilst RT-QuIC has demonstrable diagnostic value it has limited prognostic value at this point in time. [ABSTRACT FROM AUTHOR]

Classical- (C-) and atypical L-type bovine spongiform encephalopathy (BSE) prions cause different pathological phenotypes in cattle brains, and the disease-associated forms of each prion protein (PrPSc) has a dissimilar biochemical signature. Bovine C-BSE prions are the causative agent of variant Creutzfeldt-Jakob disease. To date, human infection with L-BSE prions has not been reported, but they can be transmitted experimentally from cows to cynomolgus monkeys (Macaca fascicularis), a non-human primate model. When transmitted to monkeys, C- and L-BSE prions induce different pathological phenotypes in the brain. However, when isolated from infected brains, the two prion proteins (PrPSc) have similar biochemical signatures (i.e., electrophoretic mobility, glycoforms, and resistance to proteinase K). Such similarities suggest the possibility that L-BSE prions alter their virulence to that of C-BSE prions during propagation in monkeys. To clarify this possibility, we conducted bioassays using inbred mice. C-BSE prions with or without propagation in monkeys were pathogenic to mice, and exhibited comparable incubation periods in secondary passage in mice. By contrast, L-BSE prions, either with or without propagation in monkeys, did not cause the disease in mice, indicating that the pathogenicity of L-BSE prions does not converge towards a C-BSE prion type in this primate model. These results suggest that, although C- and L-BSE prions propagated in cynomolgus monkeys exhibit similar biochemical PrPSc signatures and consist of the monkey amino acid sequence, the two prions maintain strain-specific conformations of PrPSc in which they encipher and retain unique pathogenic traits. [ABSTRACT FROM AUTHOR]

Longitudinal studies of chronic wasting disease (CWD) in the native host have provided considerable understanding of how this prion disease continues to efficiently spread among cervid species. These studies entail great cost in animal, time and financial support. A variety of methods have emerged including transgenic mouse bioassay, western blot, enzyme-linked immunoassay (ELISA), immunohistochemistry (IHC), serial protein misfolding cyclic amplification (sPMCA) and real time quaking-induced conversion (RT-QuIC), that deepen our understanding of this and other protein misfolding disorders. To further characterize an inoculum source used for ongoing CWD studies and to determine how the readouts from each of these assays compare, we assayed a CWD-positive brain pool homogenate (CBP6) and a mouse dilutional bioassay of this homogenate using the above detection methods. We demonstrate that: (i) amplification assays enhanced detection of amyloid seeding activity in the CWD+ cervid brain pool to levels beyond mouse LD50, (ii) conventional detection methods (IHC and western blot) performed well in identifying the presence of PrPSc in terminal brain tissue yet lack sufficient detection sensitivity to identify all CWD-infected mice, and (iii) the incorporation of amplification assays enhanced detection of CWD-infected mice near the LD50. This cross-platform analysis provides a basis to calibrate the relative sensitivities of CWD detection assays. [ABSTRACT FROM AUTHOR]

Neuroinflammation and neurodegeneration are common during prion infection, but the mechanisms that underlie these pathological features are not well understood. Several components of innate immunity, such as Toll-like receptor (TLR) 4 and Complement C1q, have been shown to influence prion disease. To identify additional components of innate immunity that might impact prion disease within the central nervous system (CNS), we screened RNA from brains of pre-clinical and clinical 22L-infected mice for alterations in genes associated with innate immunity. Transcription of several genes encoding damage-associated molecular pattern (DAMP) proteins and receptors were increased in the brains of prion-infected mice. To investigate the role of some of these proteins in prion disease of the CNS, we infected mice deficient in DAMP receptor genes Tlr2, C3ar1, and C5ar1 with 22L scrapie. Elimination of TLR2 accelerated disease by a median of 10 days, while lack of C3aR1 or C5aR1 had no effect on disease tempo. Histopathologically, all knockout mouse strains tested were similar to infected control mice in gliosis, vacuolation, and PrPSc deposition. Analysis of proinflammatory markers in the brains of infected knockout mice indicated only a few alterations in gene expression suggesting that C5aR1 and TLR2 signaling did not act synergistically in the brains of prion-infected mice. These results indicate that signaling through TLR2 confers partial neuroprotection during prion infection. [ABSTRACT FROM AUTHOR]

Scrapie is a naturally occurring transmissible spongiform encephalopathy of sheep and goats. This fatal neurodegenerative disease is caused by misfolding of the cellular prion protein to pathogenic β-rich conformers (PrPSc) that accumulate in higher order structures of the brain and other tissues. This conversion has been used for in vitro assays including serial protein misfolding amplification and real-time quaking induced conversion (RT-QuIC). RT-QuIC can be used for the detection of prions and for strain discrimination in a variety of biological tissues from humans and animals. In this study, we evaluated how PrPSc isolated from sheep of different genotypes after inoculation with the scrapie agent influence the fibril formation in vitro using RT-QuIC. We found that reaction mixtures seeded with PrPSc from genotype VRQ/VRQ sheep brains have better conversion efficiency with 132M elk substrate compared to reactions seeded with PrPSc from the brains of sheep with the ARQ/ARQ genotype no matter which strain of scrapie was used to seed the reactions. We also inoculated transgenic mice expressing 132M elk PRNP (Tg12) with the scrapie agent from different genotypes of sheep to compare with our RT-QuIC results. The bioassays support the data showing a significantly shorter incubation period for inoculum from VRQ/VRQ sheep when compared to inoculum from ARQ/ARQ sheep. Thus, we conclude that the genotype of both source and recipient can strongly influence transmission. [ABSTRACT FROM AUTHOR]

The prion hypothesis proposes a causal relationship between the misfolded prion protein (PrPSc) molecular entity and the disease transmissible spongiform encephalopathy (TSE). Variations in the conformation of PrPSc are associated with different forms of TSE and different risks to animal and human health. Since the discovery of atypical forms of bovine spongiform encephalopathy (BSE) in 2003, scientists have progressed the molecular characterisation of the associated PrPSc in order to better understand these risks, both in cattle as the natural host and following experimental transmission to other species. Here we report the development of a mass spectrometry based assay for molecular characterisation of bovine proteinase K (PK) treated PrPSc (PrPres) by quantitative identification of its N-terminal amino acid profiles (N-TAAPs) and tryptic peptides. We have applied the assay to classical, H-type and L-type BSE prions purified from cattle, transgenic (Tg) mice expressing the bovine (Tg110 and Tg1896) or ovine (TgEM16) prion protein gene, and sheep brain. We determined that, for classical BSE in cattle, the G96 N-terminal cleavage site dominated, while the range of cleavage sites was wider following transmission to Tg mice and sheep. For L-BSE in cattle and Tg bovinised mice, a C-terminal shift was identified in the N-TAAP distribution compared to classical BSE, consistent with observations by Western blot (WB). For L-BSE transmitted to sheep, both N-TAAP and tryptic peptide profiles were found to be changed compared to cattle, but less so following transmission to Tg ovinised mice. Relative abundances of aglycosyl peptides were found to be significantly different between the atypical BSE forms in cattle as well as in other hosts. The enhanced resolution provided by molecular analysis of PrPres using mass spectrometry has improved insight into the molecular changes following transmission of atypical BSE to other species. [ABSTRACT FROM AUTHOR]

The polymorphisms of the prion protein (PRNP) gene, which encodes normal prion proteins (PrP), are known to be involved in the susceptibility of prion diseases. The prion-like protein (Doppel) gene (PRND) is the paralog of the PRNP gene and is closely located downstream of the PRNP gene. In addition, the polymorphisms of PRND correlate with disease susceptibility in several animals. We analyzed the genotype and allele frequencies of PRND polymorphisms in 246 Korean native black goats and found a total of six single nucleotide polymorphisms (SNPs) with one novel SNP, c.99C>T. We observed linkage disequilibrium (LD) within and between loci. PRND c.28T>C, c.151A>G, and c.385G>C and PRND c.65C>T and c.286G>A were in perfect LD and we have reported for the first time strong LD between PRND and PRNP or prion-related protein gene (PRNT) loci. Specifically, between the PRND c.28T>C, c.151A>G and c.385G>C and the PRNP codon 143, PRND c.99C>T and the PRNP codon 102 or PRND SNPs (c.28T>C, c.151A>G and c.385G>C) and PRNT SNP (c.321T>C). Furthermore, we confirmed that the genotype distribution of the PRNP p.His143Arg was significantly different according to that of the PRND c.28T>C (P < 0.0001). Finally, using PolyPhen-2 and PROVEAN, we predicted that two non-synonymous SNPs, c.65C>T and c.286G>A, in the PRND gene can have a detrimental effect on Doppel. To the best of our knowledge, this is the first report of genetic characteristics of the PRND gene in Korean native black goats. [ABSTRACT FROM AUTHOR]

The risk of classical scrapie transmission in small ruminants is highest during the neonatal period with the placenta recognized as a significant source of infection. Milk has also been identified as a source of scrapie with sheep-to-sheep transmission occurring after neonatal consumption of as little as 1–2 liters of milk; concurrent mastitis due to small ruminant lentivirus (SRLV) infection may be associated with increased scrapie transmission via milk in sheep. In contrast, goat-to-sheep transmission has been documented only after prolonged consumption of >30 liters of milk. The goal of the current study was to assess transmission of scrapie to goat kids and lambs following low volume, short duration consumption of milk from infected goats. Milk from two does (female goats) with pre-clinical scrapie was fed to four goat kids (≤4.5 L each) and four lambs (~3.7 L each) beginning ~24 hours after birth. Scrapie transmission was detected in three sheep as early as 18 months post inoculation; transmission was also detected in two goats but not until postmortem analyses at 33 months post inoculation. Each milk donor goat also had naturally-acquired infection with SRLV. Different degrees of lymphohistiocytic inflammation and PrPSc accumulation were observed in mammary gland tissues of the donors, which appeared to associate with transmission of scrapie via milk. Thus, similar to the risks of milk transmission of scrapie from sheep, even limited exposure to milk from goats can pose significant risk for scrapie transmission to both goat kids and lambs. [ABSTRACT FROM AUTHOR]

Bovine spongiform encephalopathy (BSE) can be efficiently transmitted to pigs via intracerebral inoculation. A clear link has been established between the consumption of products of bovine origin contaminated with the BSE agent and the development of variant Creutzfeldt-Jakob disease in humans. Small ruminants can also naturally develop BSE, and sheep-adapted BSE (Sh-BSE) propagates more efficiently than cattle BSE in pigs and in mouse models expressing porcine prion protein. In addition, Sh-BSE shows greater efficiency of transmission to human models than original cow BSE. While infectivity and/or abnormal PrP accumulation have been reported in the central nervous system in BSE-infected pigs, the ability of the agent to replicate in peripheral tissues has not been fully investigated. We previously characterized the presence of prions in a panel of tissues collected at the clinical stage of disease from pigs experimentally infected with Sh-BSE. Western blot revealed low levels of PrPres accumulation in lymphoid tissues, nerves, and skeletal muscles from 4 of the 5 animals analysed. Using protein misfolding cyclic amplification (PMCA), which we found to be 6 log fold more sensitive than direct WB for the detection of pig BSE, we confirmed the presence of the Sh-BSE agent in lymphoid organs, nerves, ileum, and striated muscles from all 5 inoculated pigs. Surprisingly, PrPres positivity was also detected in white blood cells from one pig using this method. The presence of infectivity in lymphoid tissues, striated muscles, and peripheral nerves was confirmed by bioassay in bovine PrP transgenic mice. These results demonstrate the ability of BSE-derived agents to replicate efficiently in various peripheral tissues in pigs. Although no prion transmission has been reported in pigs following oral BSE challenge, our data support the continuation of the Feed Ban measure implemented to prevent entry of the BSE agent into the feed chain. [ABSTRACT FROM AUTHOR]

Polymorphisms at PRNP gene locus have been associated with resistance against classical scrapie in goats. Genetic selection on this gene within appropriate breeding programs may contribute to the control of the disease. The present study characterized the genetic profile of codons 146, 211 and 222 in three dairy goat breeds in Greece. A total of 766 dairy goats from seven farms were used. Animals belonged to two indigenous Greek, Eghoria (n = 264) and Skopelos (n = 287) and a foreign breed, Damascus (n = 215). Genomic DNA was extracted from blood samples from individual animals. Polymorphisms were detected in these codons using Real-Time PCR analysis and four different Custom TaqMan® SNP Genotyping Assays. Genotypic, allelic and haplotypic frequencies were calculated based on individual animal genotypes. Chi-square tests were used to examine Hardy-Weinberg equilibrium state and compare genotypic distribution across breeds. Genetic distances among the three breeds, and between these and 30 breeds reared in other countries were estimated based on haplotypic frequencies using fixation index FST with Arlequin v3.1 software; a Neighbor-Joining tree was created using PHYLIP package v3.695. Level of statistical significance was set at P = 0.01. All scrapie resistance-associated alleles (146S, 146D, 211Q and 222K) were detected in the studied population. Significant frequency differences were observed between the indigenous Greek and Damascus breeds. Alleles 222K and 146S had the highest frequency in the two indigenous and the Damascus breed, respectively (ca. 6.0%). The studied breeds shared similar haplotypic frequencies with most South Italian and Turkish breeds but differed significantly from North-Western European, Far East and some USA goat breeds. Results suggest there is adequate variation in the PRNP gene locus to support breeding programs for enhanced scrapie resistance in goats reared in Greece. Genetic comparisons among goat breeds indicate that separate breeding programs should apply to the two indigenous and the imported Damascus breeds. [ABSTRACT FROM AUTHOR]

Monitoring of small ruminants for transmissible spongiform encephalopathies (TSEs) has recently become more relevant after two natural scrapie suspected cases of goats were found to be positive for classical BSE (C-BSE). C-BSE probably established itself in this species unrecognized, undermining disease control measures. This opens the possibility that TSEs in goats may remain an animal source for human prion diseases. Currently, there are no data regarding the natural presence of the atypical BSE in caprines. Here we report that C-BSE and L-type atypical BSE (L-BSE) isolates from bovine species are intracerebrally transmissible to goats, with a 100% attack rate and a significantly shorter incubation period and survival time after C-BSE than after L-BSE experimental infection, suggesting a lower species barrier for classical agentin goat. All animals showed nearly the same clinical features of disease characterized by skin lesions, including broken hair and alopecia, and abnormal mental status. Histology and immunohistochemistry showed several differences between C-BSE and L-BSE infection, allowing discrimination between the two different strains. The lymphoreticular involvement we observed in the C-BSE positive goats argues in favour of a peripheral distribution of PrPSc similar to classical scrapie. Western blot and other currently approved screening tests detected both strains in the goats and were able to classify negative control animals. These data demonstrate that active surveillance of small ruminants, as applied to fallen stock and/or healthy slaughter populations in European countries, is able to correctly identify and classify classical and L-BSE and ultimately protect public health. [ABSTRACT FROM AUTHOR]

The cellular prion protein (PrPC) is implicated in neuroprotective signaling and neurotoxic pathways in both prion diseases and Alzheimer’s disease (AD). Specifically, the intrinsically disordered N-terminal domain (N-PrP) has been shown to interact with neurotoxic ligands, such as Aβ and Scrapie prion protein (PrPSc), and to be crucial for the neuroprotective activity of PrPC. To gain further insight into cellular pathways tied to PrP, we analyzed the brain interactome of N-PrP. As a novel approach employing recombinantly expressed PrP and intein-mediated protein ligation, we used N-PrP covalently coupled to beads as a bait for affinity purification. N-PrP beads were incubated with human AD or control brain lysates. N-PrP binding partners were then identified by electrospray ionization tandem mass spectrometry (nano ESI-MS/MS). In addition to newly identified proteins we found many previously described PrP interactors, indicating a crucial role of the intrinsically disordered part of PrP in mediating protein interactions. Moreover, some interactors were found only in either non-AD or AD brain, suggesting aberrant PrPC interactions in the pathogenesis of AD. [ABSTRACT FROM AUTHOR]

Chronic wasting disease (CWD) is a fatal neurodegenerative disease of deer, elk, moose, and reindeer (cervids) caused by misfolded prion proteins. The disease has been reported across North America and recently discovered in northern Europe. Transmission of CWD in wild cervid populations can occur through environmental routes, but limited ability to detect prions in environmental samples has prevented the identification of potential transmission “hot spots”. We establish widespread CWD prion contamination of mineral licks used by free-ranging cervids in an enzootic area in Wisconsin, USA. We show mineral licks can serve as reservoirs of CWD prions and thus facilitate disease transmission. Furthermore, mineral licks attract livestock and other wildlife that also obtain mineral nutrients via soil and water consumption. Exposure to CWD prions at mineral licks provides potential for cross-species transmission to wildlife, domestic animals, and humans. Managing deer use of mineral licks warrants further consideration to help control outbreaks of CWD. [ABSTRACT FROM AUTHOR]

Scrapie is a transmissible spongiform encephalopathy in sheep and an example of a disease that may be controlled through breeding for disease resistance. Member states of the European Union have introduced strategies for breeding against scrapie based on the selection of genetically resistant breeding rams. An ambitious strategy adopted in The Netherlands consisted of selecting resistant rams for breeding throughout both breeding and production sectors. Mathematical modelling of the effect of a breeding program on the spreading capacity of scrapie in a national flock is needed for making assessments on how long a breeding strategy needs to be maintained to achieve disease control. Here we describe such a model applied to the Dutch situation, with the use of data on the genetic content of the Dutch sheep population as well as on scrapie occurrence in this population. We show that the time needed for obtaining scrapie control depends crucially on two parameters measuring sheep population structure: the between-flock heterogeneity in genotype frequencies, and the heterogeneity of mixing (contact rates) between sheep flocks. Estimating the first parameter from Dutch genetic survey data and assuming scenario values for the second one, enables model prediction of the time needed to achieve scrapie control in The Netherlands. [ABSTRACT FROM AUTHOR]

Prion diseases have been linked to impaired copper homeostasis and copper induced-oxidative damage to the brain. Divalent metal ions, such as Cu2+ and Zn2+, bind to cellular prion protein (PrPC) at octapeptide repeat (OR) and non-OR sites within the N-terminal half of the protein but information on the impact of such binding on conversion to the misfolded isoform often derives from studies using either OR and non-OR peptides or bacterially-expressed recombinant PrP. Here we created new transgenic mouse lines expressing PrP with disrupted copper binding sites within all four histidine-containing OR's (sites 1–4, H60G, H68G, H76G, H84G, "TetraH>G" allele) or at site 5 (composed of residues His-95 and His-110; "H95G" allele) and monitored the formation of misfolded PrP in vivo. Novel transgenic mice expressing PrP(TetraH>G) at levels comparable to wild-type (wt) controls were susceptible to mouse-adapted scrapie strain RML but showed significantly prolonged incubation times. In contrast, amino acid replacement at residue 95 accelerated disease progression in corresponding PrP(H95G) mice. Neuropathological lesions in terminally ill transgenic mice were similar to scrapie-infected wt controls, but less severe. The pattern of PrPSc deposition, however, was not synaptic as seen in wt animals, but instead dense globular plaque-like accumulations of PrPSc in TgPrP(TetraH>G) mice and diffuse PrPSc deposition in (TgPrP(H95G) mice), were observed throughout all brain sections. We conclude that OR and site 5 histidine substitutions have divergent phenotypic impacts and that cis interactions between the OR region and the site 5 region modulate pathogenic outcomes by affecting the PrP globular domain. [ABSTRACT FROM AUTHOR]

Humic substances (HS) are the largest constituent of soil organic matter and are considered as a key component of the terrestrial ecosystem. HS may facilitate the transport of organic and inorganic molecules, as well as the sorption interactions with environmentally relevant proteins such as prions. Prions enter the environment through shedding from live hosts, facilitating a sustained incidence of animal prion diseases such as Chronic Wasting Disease and scrapie in cervid and ovine populations, respectively. Changes in prion structure upon environmental exposure may be significant as they can affect prion infectivity and disease pathology. Despite its relevance, the mechanisms of prion interaction with HS are still not completely understood. The goal of this work is to advance a structural-level picture of the encapsulation of recombinant, non-infectious, prion protein (PrP) into different natural HS. We observed that PrP precipitation upon addition of HS is mainly driven by a mechanism of “salting-out” whereby PrP molecules are rapidly removed from the solution and aggregate in insoluble adducts with humic molecules. Importantly, this process does not alter the protein folding since insoluble PrP retains its α-helical content when in complex with HS. The observed ability of HS to promote PrP insolubilization without altering its secondary structure may have potential relevance in the context of “prion ecology”. These results suggest that soil organic matter interacts with prions possibly without altering the protein structures. This may facilitate prions preservation from biotic and abiotic degradation leading to their accumulation in the environment. [ABSTRACT FROM AUTHOR]

Chronic wasting disease (CWD) is a fatal transmissible spongiform encephalopathy affecting white-tailed deer (Odocoileus virginianus), mule deer (Odocoileus hemionus), Rocky Mountain elk (Cervus elaphus nelsoni), and moose (Alces alces shirasi) in North America. In southeastern Wyoming average annual CWD prevalence in mule deer exceeds 20% and appears to contribute to regional population declines. We determined the effect of CWD on mule deer demography using age-specific, female-only, CWD transition matrix models to estimate the population growth rate (λ). Mule deer were captured from 2010–2014 in southern Converse County Wyoming, USA. Captured adult (≥ 1.5 years old) deer were tested ante-mortem for CWD using tonsil biopsies and monitored using radio telemetry. Mean annual survival rates of CWD-negative and CWD-positive deer were 0.76 and 0.32, respectively. Pregnancy and fawn recruitment were not observed to be influenced by CWD. We estimated λ = 0.79, indicating an annual population decline of 21% under current CWD prevalence levels. A model derived from the demography of only CWD-negative individuals yielded; λ = 1.00, indicating a stable population if CWD were absent. These findings support CWD as a significant contributor to mule deer population decline. Chronic wasting disease is difficult or impossible to eradicate with current tools, given significant environmental contamination, and at present our best recommendation for control of this disease is to minimize spread to new areas and naïve cervid populations. [ABSTRACT FROM AUTHOR]

The agent responsible for prion diseases is a misfolded form of a normal protein (PrPC). The prion hypothesis stipulates that PrPC must be present for the disease to manifest. Cervid populations across the world are infected with chronic wasting disease, a horizontally-transmissible prion disease that is likely spread via oral exposure to infectious prions (PrPCWD). Though PrPCWD has been identified in many tissues, there has been little effort to characterize the overall PrPC expression in cervids and its relationship to PrPCWD accumulation. We used immunohistochemistry (IHC), western blot and enzyme-linked immunosorbent assay to describe PrPC expression in naïve white-tailed deer. We used real-time, quaking-induced conversion (RT-QuIC) to detect prion seeding activity in CWD-infected deer. We assessed tissues comprising the alimentary tract, alimentary-associated lymphoid tissue and systemic lymphoid tissue from 5 naïve deer. PrPC was expressed in all tissues, though expression was often very low compared to the level in the CNS. IHC identified specific cell types wherein PrPC expression is very high. To compare the distribution of PrPC to PrPCWD, we examined 5 deer with advanced CWD infection. Using RT-QuIC, we detected prion seeding activity in all 21 tissues. In 3 subclinical deer sacrificed 4 months post-inoculation, we detected PrPCWD consistently in alimentary-associated lymphoid tissue, irregularly in alimentary tract tissues, and not at all in the brain. Contrary to our hypothesis that PrPC levels dictate prion accumulation, PrPC expression was higher in the lower gastrointestinal tissues than in the alimentary-associated lymphoid system and was higher in salivary glands than in the oropharyngeal lymphoid tissue. These data suggest that PrPC expression is not the sole driver of prion accumulation and that alimentary tract tissues accumulate prions before centrifugal spread from the brain occurs. [ABSTRACT FROM AUTHOR]

In our previous study, we demonstrated the propagation of mouse-passaged scrapie isolates with long incubation periods (L-type) derived from natural Japanese sheep scrapie cases in murine hypothalamic GT1-7 cells, along with disease-associated prion protein (PrPSc) accumulation. We here analyzed the susceptibility of GT1-7 cells to scrapie prions by exposure to infected mouse brains at different passages, following interspecies transmission. Wild-type mice challenged with a natural sheep scrapie case (Kanagawa) exhibited heterogeneity of transmitted scrapie prions in early passages, and this mixed population converged upon one with a short incubation period (S-type) following subsequent passages. However, when GT1-7 cells were challenged with these heterologous samples, L-type prions became dominant. This study demonstrated that the susceptibility of GT1-7 cells to L-type prions was at least 105 times higher than that to S-type prions and that L-type prion-specific biological characteristics remained unchanged after serial passages in GT1-7 cells. This suggests that a GT1-7 cell culture model would be more useful for the economical and stable amplification of L-type prions at the laboratory level. Furthermore, this cell culture model might be used to selectively propagate L-type scrapie prions from a mixed prion population. [ABSTRACT FROM AUTHOR]

Prion diseases are neurodegenerative conditions caused by misfolding of the prion protein, leading to conspicuous neuronal loss and intense microgliosis. Recent experimental evidence point towards a protective role of microglia against prion-induced neurodegeneration, possibly through elimination of prion-containing apoptotic bodies. The molecular mechanisms by which microglia recognize and eliminate apoptotic cells in the context of prion diseases are poorly defined. Here we investigated the possible involvement of signal regulatory protein α (SIRPα), a key modulator of host cell phagocytosis; SIRPα is encoded by the Sirpa gene that is genetically linked to the prion gene Prnp. We found that Sirpa transcripts are highly enriched in microglia cells within the brain. However, Sirpa mRNA levels were essentially unaltered during the course of experimental prion disease despite upregulation of other microglia-enriched transcripts. To study the involvement of SIRPα in prion pathogenesis in vivo, mice expressing a truncated SIRPα protein unable to inhibit phagocytosis were inoculated with rodent-adapted scrapie prions of the 22L strain. Homozygous and heterozygous Sirpa mutants and wild-type mice experienced similar incubation times after inoculation with either of two doses of 22L prions. Moreover, the extent of neuronal loss, microgliosis and abnormal prion protein accumulation was not significantly affected by Sirpa genotypes. Collectively, these data indicate that SIRPα-mediated phagocytosis is not a major determinant in prion disease pathogenesis. It will be important to search for additional candidates mediating prion phagocytosis, as this mechanism may represent an important target of antiprion therapies. [ABSTRACT FROM AUTHOR]

Animal social behaviour can have important effects on the long-term dynamics of diseases. In particular, preferential spatial relationships between individuals can lead to differences in the rates of disease spread within a population. We examined the concurrent influence of genetic relatedness, sex, age, home range overlap, time of year, and prion disease status on proximal associations of adult Rocky Mountain mule deer (Odocoileus hemionus hemionus) in a chronic wasting disease endemic area. We also quantified the temporal stability of these associations across different sex, age, and disease status classes. We used three years of high frequency telemetry data from 74 individuals to record encounters within 25 m of each other, and to calculate seasonal home range overlap measured by volume of intersection (VI). The strength of pairwise spatial association between adult mule deer was independent of genetic relatedness, age and disease status. Seasonal variation in association strength was not consistent across years, perhaps due to annual changes in weather conditions. The influence of home range overlap on association strength varied seasonally, whereby associations were stronger in pre-rut and fawning than in the rest of the seasons. The sexes of individuals also interacted with both VI and season. At increasing levels of VI, associations were stronger between females than between males and between females and males. The strongest associations in pre-rut were between males, while the strongest in rut were between females and males. The temporal stability of associations was markedly dependant on the sex and the diagnosis of the associating pair. Our findings highlight the importance of considering concurrent effects of biological and environmental factors when seeking to understand the role of social preference in behavioural ecology and disease spread. Applying this knowledge in epidemiological modelling will shed light on the dynamics of disease transmission among mule deer. [ABSTRACT FROM AUTHOR]

The transmission of classical bovine spongiform encephalopathy (C-BSE) through contaminated meat product consumption is responsible for variant Creutzfeldt-Jakob disease (vCJD) in humans. More recent and atypical forms of BSE (L-BSE and H-BSE) have been identified in cattle since the C-BSE epidemic. Their low incidence and advanced age of onset are compatible with a sporadic origin, as are most cases of Creutzfeldt-Jakob disease (CJD) in humans. Transmissions studies in primates and transgenic mice expressing a human prion protein (PrP) indicated that atypical forms of BSE may be associated with a higher zoonotic potential than classical BSE, and require particular attention for public health. Recently, methods designed to amplify misfolded forms of PrP have emerged as promising tools to detect prion strains and to study their diversity. Here, we validated real-time quaking-induced conversion assay for the discrimination of atypical and classical BSE strains using a large series of bovine samples encompassing all the atypical BSE cases detected by the French Centre of Reference during 10 years of exhaustive active surveillance. We obtained a 100% sensitivity and specificity for atypical BSE detection. In addition, the assay was able to discriminate atypical and classical BSE in non-human primates, and also sporadic CJD and vCJD in humans. The RT-QuIC assay appears as a practical means for a reliable detection of atypical BSE strains in a homologous or heterologous PrP context. [ABSTRACT FROM AUTHOR]

Prions are amyloid-forming proteins that cause transmissible spongiform encephalopathies through a process involving conversion from the normal cellular prion protein to the pathogenic misfolded conformation (PrPSc). This conversion has been used for in vitro assays including serial protein misfolding amplification and real-time quaking induced conversion (RT-QuIC). RT-QuIC can be used for the detection of prions in a variety of biological tissues from humans and animals. Extensive work has been done to demonstrate that RT-QuIC is a rapid, specific, and highly sensitive prion detection assay. RT-QuIC uses recombinant prion protein to detect minute amounts of PrPSc. RT-QuIC has been successfully used to detect PrPSc from different prion diseases with a variety of substrates including hamster, human, sheep, bank vole, bovine and chimeric forms of prion protein. However, recombinant bovine prion protein has not been used to detect transmissible mink encephalopathy (TME) or to differentiate types of bovine spongiform encephalopathy (BSE) in samples from cattle. We evaluated whether PrPSc from TME and BSE infected cattle can be detected with RT-QuIC using recombinant bovine prion proteins, and optimized the reaction conditions to specifically detect cattle TME and to discriminate between classical and atypical BSE by conversion efficiency. We also found that substrate composed of the disease associated E211K mutant protein can be effective for the detection of TME in cattle and that wild type prion protein appears to be a practical substrate to discriminate between the different types of BSEs. [ABSTRACT FROM AUTHOR]

Resistance to proteolytic digestion has long been considered a defining trait of prions in tissues of organisms suffering from transmissible spongiform encephalopathies. Detection of proteinase K-resistant prion protein (PrPSc) still represents the diagnostic gold standard for prion diseases in humans, sheep and cattle. However, it has become increasingly apparent that the accumulation of PrPSc does not always accompany prion infections: high titers of prion infectivity can be reached also in the absence of protease resistant PrPSc. Here, we describe a structural basis for the phenomenon of protease-sensitive prion infectivity. We studied the effect on proteinase K (PK) resistance of the amino acid substitution Y169F, which removes a single oxygen atom from the β2–α2 loop of the cellular prion protein (PrPC). When infected with RML or the 263K strain of prions, transgenic mice lacking wild-type (wt) PrPC but expressing MoPrP169F generated prion infectivity at levels comparable to wt mice. The newly generated MoPrP169F prions were biologically indistinguishable from those recovered from prion-infected wt mice, and elicited similar pathologies in vivo. Surprisingly, MoPrP169F prions showed greatly reduced PK resistance and density gradient analyses showed a significant reduction in high-density aggregates. Passage of MoPrP169F prions into mice expressing wt MoPrP led to full recovery of protease resistance, indicating that no strain shift had taken place. We conclude that a subtle structural variation in the β2–α2 loop of PrPC affects the sensitivity of PrPSc to protease but does not impact prion replication and infectivity. With these findings a specific structural feature of PrPC can be linked to a physicochemical property of the corresponding PrPSc. [ABSTRACT FROM AUTHOR]

Prions are unique infectious agents, consisting solely of abnormally-folded prion protein (PrPSc). However, they possess virus-like features, including strain diversity, the ability to adapt to new hosts and to be altered evolutionarily. Because prions lack genetic material (DNA and RNA), these biological phenomena have been attributed to the structural properties of PrPSc. Therefore, many structural models of the structure of PrPSc have been proposed based on the limited structural information available, regardless of the incompatibility with high-resolution structural analysis. Recently hypothesized models consist solely of β-sheets and intervening loops/kinks; i.e. parallel in-register β-sheet and β-solenoid models. Owing to the relative simplicity of these structural models of PrPSc, we hypothesized that numerical conversion of the primary structures with a relevant algorithm would enable quantitative comparison between PrPs of distinct primary structures. We therefore used the theoretical values of β-sheet (Pβ) and random-coil (Pc) propensity calculated by secondary structure prediction with a neural network, to analyze interspecies transmission of prions. By reviewing experiments in the literature, we ascertained the biological relevance of Pβ and Pc and found that these classical parameters surprisingly carry substantial information of amyloid structures. We also demonstrated how these parameters could aid in interpreting and explaining phenomena in interspecies transmissions. Our approach can lead to the development of a versatile tool for investigating not only prions but also other amyloids. [ABSTRACT FROM AUTHOR]

Misfolding of the cellular prion protein (PrPC) into the scrapie prion protein (PrPSc) results in progressive, fatal, transmissible neurodegenerative conditions termed prion diseases. Experimental and epidemiological evidence point toward a protracted, clinically silent phase in prion diseases, yet there is no diagnostic test capable of identifying asymptomatic individuals incubating prions. In an effort to identify early biomarkers of prion diseases, we have compared global transcriptional profiles in brains from pre-symptomatic prion-infected mice and controls. We identified Cst7, which encodes cystatin F, as the most strongly upregulated transcript in this model. Early and robust upregulation of Cst7 mRNA levels and of its cognate protein was validated in additional mouse models of prion disease. Surprisingly, we found no significant increase in cystatin F levels in both cerebrospinal fluid or brain parenchyma of patients with Creutzfeldt-Jakob disease compared to Alzheimer’s disease or non-demented controls. Our results validate cystatin F as a useful biomarker of early pathogenesis in experimental models of prion disease, and point to unexpected species-specific differences in the transcriptional responses to prion infections. [ABSTRACT FROM AUTHOR]

The propensity of cellular prion protein to aggregation is reputed essential for the initiation of the amyloid cascade that ultimately lead to the accumulation of neurotoxic aggregates. In this paper, we extended and applied an already reported computational workflow [Proteins 2015; 83: 1751–1765] to elucidate in details the aggregation propensity of PrP protein systems including wild type, wild type treated at different [Ca2+] and E200K mutant. The application of the computational procedure to two segments of PrPC, i.e. 125–228 and 120–231, allowed to emphasize how the inclusion of complete C-terminus and last portion (120–126) of the neurotoxic segment 106–126 may be crucial to unveil significant and unexpected interaction properties. Indeed, the anchoring of N-terminus on H2 domain detected in the wild type resulted to be disrupted upon either E200K mutation or Ca2+ binding, and to unbury hydrophobic spots on the PrPC surface. A peculiar dinuclear Ca2+ binding motif formed by the C-terminus and the S2-H2 loop was detected for [Ca2+] > 5 mM and showed similarities with binding motifs retraced in other protein systems, thus suggesting a possible functional meaning for its formation. Therefore, we potentiated the computational procedure by including a tool that clusterize the minima of molecular interaction fields of a proteinand delimit the regions of space with higher hydrophobic or higher hydrophilic character, hence, more likely involved in the self-assembly process. Plausible models for the self-assembly of either the E200K mutated or Ca2+-bound PrPC were sketched and discussed. The present investigation provides for structure-based information and new prompts that may represent a starting point for future experimental or computational works on the PrPC aggregation. [ABSTRACT FROM AUTHOR]

Prion diseases are infectious and fatal neurodegenerative diseases which require the cellular prion protein, PrPC, for development of diseases. The current study shows that the PrPC augments infectivity and plaque formation of a mouse endogenous retrovirus, MuLV. We have established four neuronal cell lines expressing mouse PrPC, PrP+/+; two express wild type PrPC (MoPrPwild) and the other two express mutant PrPC (MoPrPmut). Infection of neuronal cells from various PrP+/+ and PrP-/- (MoPrPKO) lines with MuLV yielded at least three times as many plaques in PrP+/+ than in PrP-/-. Furthermore, among the four PrP+/+ lines, one mutant line, P101L, had at least 2.5 times as many plaques as the other three PrP+/+ lines. Plaques in P101L were four times larger than those in other PrP+/+ lines. Colocalization of PrP and CAgag was seen in MuLV-infected PrP+/+ cells. In the PrP-MuLV interaction, the involvement of galectin-3 and -6 was observed by immunoprecipitation with antibody to PrPC. These results suggest that PrPC combined with galectin-3 and -6 can act as a receptor for MuLV. P101L, the disease form of mutant PrPC results suggest the genetic mutant form of PrPC may be more susceptible to viral infection. [ABSTRACT FROM AUTHOR]

Chronic wasting disease (CWD) is a fatal prion disease of wild and captive cervids in North America. Prions are infectious agents composed of a misfolded version of a host-encoded protein, termed PrPSc. Infected cervids excrete and secrete prions, contributing to lateral transmission. Geographical distribution is expanding and case numbers in wild cervids are increasing. Recently, the first European cases of CWD have been reported in a wild reindeer and two moose from Norway. Therefore, methods to detect the infection early in the incubation time using easily available samples are desirable to facilitate effective disease management. We have adapted the real-time quaking induced conversion (RT-QuIC) assay, a sensitive in vitro prion amplification method, for pre-clinical detection of prion seeding activity in elk feces. Testing fecal samples from orally inoculated elk taken at various time points post infection revealed early shedding and detectable prion seeding activity throughout the disease course. Early shedding was also found in two elk encoding a PrP genotype associated with reduced susceptibility for CWD. In summary, we suggest that detection of CWD prions in feces by RT-QuIC may become a useful tool to support CWD surveillance in wild and captive cervids. The finding of early shedding independent of the elk’s prion protein genotype raises the question whether prolonged survival is beneficial, considering accumulation of environmental prions and its contribution to CWD transmission upon extended duration of shedding. [ABSTRACT FROM AUTHOR]

White-tailed deer are a culturally and economically important game species in North America, especially in South Texas. The recent discovery of chronic wasting disease (CWD) in captive deer facilities in Texas has increased concern about the potential emergence of CWD in free-ranging deer. The concern is exacerbated because much of the South Texas region is a semi-arid environment with variable rainfall, where precipitation is strongly correlated with fawn recruitment. Further, the marginally productive rangelands, in combination with erratic fawn recruitment, results in populations that are frequently density-independent, and thus sensitive to additive mortality. It is unknown how a deer population in semi-arid regions would respond to the presence of CWD. We used long-term empirical datasets from a lightly harvested (2% annual harvest) population in conjunction with 3 prevalence growth rates from CWD afflicted areas (0.26%, 0.83%, and 2.3% increases per year) via a multi-stage partially deterministic model to simulate a deer population for 25 years under four scenarios: 1) without CWD and without harvest, 2) with CWD and without harvest, 3) with CWD and male harvest only, and 4) with CWD and harvest of both sexes. The modeled populations without CWD and without harvest averaged a 1.43% annual increase over 25 years; incorporation of 2% annual harvest of both sexes resulted in a stable population. The model with slowest CWD prevalence rate growth (0.26% annually) without harvest resulted in stable populations but the addition of 1% harvest resulted in population declines. Further, the male age structure in CWD models became skewed to younger age classes. We incorporated fawn:doe ratios from three CWD afflicted areas in Wisconsin and Wyoming into the model with 0.26% annual increase in prevalence and populations did not begin to decline until ~10%, ~16%, and ~26% of deer were harvested annually. Deer populations in variable environments rely on high adult survivorship to buffer the low and erratic fawn recruitment rates. The increase in additive mortality rates for adults via CWD negatively impacted simulated population trends to the extent that hunter opportunity would be greatly reduced. Our results improve understanding of the potential influences of CWD on deer populations in semi-arid environments with implications for deer managers, disease ecologists, and policy makers. [ABSTRACT FROM AUTHOR]

Prions are the infectious agents causing transmissible spongiform encephalopathies (TSE), progressive, inexorably lethal neurological diseases. Antibodies targeting the globular domain (GD) of the cellular prion protein PrPC trigger a neurotoxic syndrome morphologically and molecularly similar to prion disease. This phenomenon raises the question whether such antibodies induce infectious prions de novo. Here we exposed cerebellar organotypic cultured slices (COCS) to the neurotoxic antibody, POM1. We then inoculated COCS homogenates into tga20 mice, which overexpress PrPC and are commonly utilized as sensitive indicators of prion infectivity. None of the mice inoculated with COCS-derived lysates developed any signs of disease, and all mice survived for at least 200 days post-inoculation. In contrast, all mice inoculated with bona fide prions succumbed to TSE after 55–95 days. Post-mortem analyses did not reveal any signs of prion pathology in mice inoculated with POM1-COCS lysates. Also, lysates from POM1-exposed COCS were unable to convert PrP by quaking. Hence, anti-GD antibodies do not catalyze the generation of prion infectivity. These data indicate that prion replication can be separated from prion toxicity, and suggest that anti-GD antibodies exert toxicity by acting downstream of prion replication. [ABSTRACT FROM AUTHOR]

Chronic wasting disease (CWD) is an invariably fatal transmissible spongiform encephalopathy of white-tailed deer, mule deer, elk, and moose. Despite a 100% fatality rate, areas of high prevalence, and increasingly expanding geographic endemic areas, little is known about the population-level effects of CWD in deer. To investigate these effects, we tested the null hypothesis that high prevalence CWD did not negatively impact white-tailed deer population sustainability. The specific objectives of the study were to monitor CWD-positive and CWD-negative white-tailed deer in a high-prevalence CWD area longitudinally via radio-telemetry and global positioning system (GPS) collars. For the two populations, we determined the following: a) demographic and disease indices, b) annual survival, and c) finite rate of population growth (λ). The CWD prevalence was higher in females (42%) than males (28.8%) and hunter harvest and clinical CWD were the most frequent causes of mortality, with CWD-positive deer over-represented in harvest and total mortalities. Survival was significantly lower for CWD-positive deer and separately by sex; CWD-positive deer were 4.5 times more likely to die annually than CWD-negative deer while bucks were 1.7 times more likely to die than does. Population λ was 0.896 (0.859–0.980), which indicated a 10.4% annual decline. We show that a chronic disease that becomes endemic in wildlife populations has the potential to be population-limiting and the strong population-level effects of CWD suggest affected populations are not sustainable at high disease prevalence under current harvest levels. [ABSTRACT FROM AUTHOR]

Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are a group of fatal neurodegenerative disorders infecting both humans and animals. Recent works have demonstrated that the soluble prion protein oligomer (PrPO), the intermediate of the conformational transformation from the host-derived cellular form (PrPC) to the disease-associated Scrapie form (PrPSc), exerts the major neurotoxicity in vitro and in vivo. Rabbits show strong resistance to TSEs, the underlying mechanism is unclear to date. It is expected that the relative TSEs-resistance of rabbits is closely associated with the unique properties of rabbit prion protein oligomer which remain to be addressed in detail. In the present work, we prepared rabbit prion protein oligomer (recRaPrPO) and human prion protein oligomer (recHuPrPO) under varied conditions, analyzed the effects of pH, NaCl concentration and incubation temperature on the oligomerization, and compared the properties of recRaPrPO and recHuPrPO. We found that several factors facilitated the formation of prion protein oligomers, including low pH, high NaCl concentration, high incubation temperature and low conformational stability of monomeric prion protein. RecRaPrPO was formed more slowly than recHuPrPO at physiological-like conditions (< 57°C, < 150 mM NaCl). Furthermore, recRaPrPO possessed higher susceptibility to proteinase K and lower cytotoxicity in vitro than recHuPrPO. These unique properties of recRaPrPO might substantially contribute to the TSEs-resistance of rabbits. Our work sheds light on the oligomerization of prion proteins and is of benefit to mechanistic understanding of TSEs-resistance of rabbits. [ABSTRACT FROM AUTHOR]

Previous investigations aimed at determining whether the mammalian prion protein actually facilitates tangible molecular aspects of either a discrete or pleiotropic functional niche have been debated, especially given the apparent absence of overt behavioral or physiological phenotypes associated with several mammalian prion gene (PRNP) knockout experiments. Moreover, a previous evaluation of PRNP knockout cattle concluded that they were normal, suggesting that the bovine prion protein is physiologically dispensable. Herein, we examined the frequency and distribution of nucleotide sequence variation within the coding regions of bovine PRNP and the adjacent Doppel (PRND) gene, a proximal paralogue to PRNP on BTA13. Evaluation of PRND variation demonstrated that the gene does not depart from a strictly neutral model of molecular evolution, and would therefore not be expected to influence tests of selection within PRNP. Collectively, our analyses confirm that intense purifying selection is indeed occurring directly on bovine PRNP, which is indicative of a protein with an important role. These results suggest that the lack of observed fitness effects may not manifest in the controlled environmental conditions used to care for and raise PRNP knockout animals. [ABSTRACT FROM AUTHOR]

Prion diseases, such as bovine spongiform encephalopathies (BSE), are transmissible neurodegenerative disorders affecting humans and a wide variety of mammals. Variant Creutzfeldt-Jakob disease (vCJD), a prion disease in humans, has been linked to exposure to BSE prions. This classical BSE (cBSE) is now rapidly disappearing as a result of appropriate measures to control animal feeding. Besides cBSE, two atypical forms (named H- and L-type BSE) have recently been described in Europe, Japan, and North America. Here we describe the first wide-spectrum microarray analysis in whole blood of atypical BSE-infected cattle. Transcriptome changes in infected animals were analyzed prior to and after the onset of clinical signs. The microarray analysis revealed gene expression changes in blood prior to the appearance of the clinical signs and during the progression of the disease. A set of 32 differentially expressed genes was found to be in common between clinical and preclinical stages and showed a very similar expression pattern in the two phases. A 22-gene signature showed an oscillating pattern of expression, being differentially expressed in the preclinical stage and then going back to control levels in the symptomatic phase. One gene, SEL1L3, was downregulated during the progression of the disease. Most of the studies performed up to date utilized various tissues, which are not suitable for a rapid analysis of infected animals and patients. Our findings suggest the intriguing possibility to take advantage of whole blood RNA transcriptional profiling for the preclinical identification of prion infection. Further, this study highlighted several pathways, such as immune response and metabolism that may play an important role in peripheral prion pathogenesis. Finally, the gene expression changes identified in the present study may be further investigated as a fingerprint for monitoring the progression of disease and for developing targeted therapeutic interventions. [ABSTRACT FROM AUTHOR]

Infectious disease dynamics are determined, to a great extent, by the social structure of the host. We evaluated sociality, or the tendency to form groups, in Rocky Mountain mule deer (Odocoileus hemionus hemionus) from a chronic wasting disease (CWD) endemic area in Saskatchewan, Canada, to better understand factors that may affect disease transmission. Using group size data collected on 365 radio-collared mule deer (2008–2013), we built a generalized linear mixed model (GLMM) to evaluate whether factors such as CWD status, season, habitat and time of day, predicted group occurrence. Then, we built another GLMM to determine factors associated with group size. Finally, we used 3 measures of group size (typical, mean and median group sizes) to quantify levels of sociality. We found that mule deer showing clinical signs of CWD were less likely to be reported in groups than clinically healthy deer after accounting for time of day, habitat, and month of observation. Mule deer groups were much more likely to occur in February and March than in July. Mixed-sex groups in early gestation were larger than any other group type in any season. Groups were largest and most likely to occur at dawn and dusk, and in open habitats, such as cropland. We discuss the implication of these results with respect to sociobiology and CWD transmission dynamics. [ABSTRACT FROM AUTHOR]

The minimum dose required to cause infection of Romney and Suffolk sheep of the ARQ/ARQ or ARQ/ARR prion protein gene genotypes following oral inoculation with Romney or Suffolk a sheep Bovine spongiform encephalopathy (BSE)-derived or cattle BSE-derived agent was investigated using doses ranging from 0.0005g to 5g. ARQ/ARQ sheep which were methionine (M) / threonine (T) heterozygous or T/T homozygous at codon 112 of the Prnp gene, dosed ARQ/ARR sheep and undosed controls did not show any evidence of infection. Within groups of susceptible sheep, the minimum effective oral dose of BSE was found to be 0.05g, with higher attack rates following inoculation with the 5g dose. Surprisingly, this study found no effect of dose on survival time suggesting a possible lack of homogeneity within the inoculum. All clinical BSE cases showed PrPd accumulation in brain; however, following cattle BSE inoculation, LRS involvement within Romney recipients was found to be significantly lower than within the Suffolk sheep inoculated group which is in agreement with previous reports. [ABSTRACT FROM AUTHOR]

Disease prioritization exercises have been used by several organizations to inform surveillance and control measures. Though most methodologies for disease prioritization are based on expert opinion, it is becoming more common to include different stakeholders in the prioritization exercise. This study was performed to compare the weighting of disease criteria, and the consequent prioritization of zoonoses, by both health professionals and students in Switzerland using a Conjoint Analysis questionnaire. The health professionals comprised public health and food safety experts, cantonal physicians and cantonal veterinarians, while the student group comprised first-year veterinary and agronomy students. Eight criteria were selected for this prioritization based on expert elicitation and literature review. These criteria, described on a 3-tiered scale, were evaluated through a choice-based Conjoint Analysis questionnaire with 25 choice tasks. Questionnaire results were analyzed to obtain importance scores (for each criterion) and mean utility values (for each criterion level), and the latter were then used to rank 16 zoonoses. While the most important criterion for both groups was “Severity of the disease in humans”, the second ranked criteria by the health professionals and students were “Economy” and “Treatment in humans”, respectively. Regarding the criterion “Control and Prevention”, health professionals tended to prioritize a disease when the control and preventive measures were described to be 95% effective, while students prioritized a disease if there were almost no control and preventive measures available. Bovine Spongiform Encephalopathy was the top-ranked disease by both groups. Health professionals and students agreed on the weighting of certain criteria such as “Severity” and “Treatment of disease in humans”, but disagreed on others such as “Economy” or “Control and Prevention”. Nonetheless, the overall disease ranking lists were similar, and these may be taken into consideration when making future decisions regarding resource allocation for disease control and prevention in Switzerland. [ABSTRACT FROM AUTHOR]

We develop a model of CWD management by nonselective deer harvest, currently the most feasible approach available for managing CWD in wild populations. We use the model to explore the effects of 6 common harvest strategies on disease prevalence and to identify potential optimal harvest policies for reducing disease prevalence without population collapse. The model includes 4 deer categories (juveniles, adult females, younger adult males, older adult males) that may be harvested at different rates, a food-based carrying capacity, which influences juvenile survival but not adult reproduction or survival, and seasonal force of infection terms for each deer category under differing frequency-dependent transmission dynamics resulting from environmental and direct contact mechanisms. Numerical experiments show that the interval of transmission coefficients β where the disease can be controlled is generally narrow and efficiency of a harvest policy to reduce disease prevalence depends crucially on the details of the disease transmission mechanism, in particular on the intensity of disease transmission to juveniles and the potential differences in the behavior of older and younger males that influence contact rates. Optimal harvest policy to minimize disease prevalence for each of the assumed transmission mechanisms is shown to depend on harvest intensity. Across mechanisms, a harvest that focuses on antlered deer, without distinguishing between age classes reduces disease prevalence most consistently, whereas distinguishing between young and older antlered deer produces higher uncertainty in the harvest effects on disease prevalence. Our results show that, despite uncertainties, a modelling approach can determine classes of harvest strategy that are most likely to be effective in combatting CWD. [ABSTRACT FROM AUTHOR]

Susceptibility to infection by prions is highly dependent on the amino acid sequence and host expression of the cellular prion protein (PrPC); however, cellular expression of a genetically susceptible PrPC is insufficient. As an example, it has been shown in cultured cells that permissive and resistant sublines derived from the same parental population often have similar expression levels of PrPC. Thus, additional cellular factors must influence susceptibility to prion infection. The aim of this study was to elucidate the factors associated with relative permissiveness and resistance to scrapie prions in cultured cells derived from a naturally affected species. Two closely related ovine microglia clones with different prion susceptibility, but no detectable differences in PrPC expression levels, were inoculated with either scrapie-positive or scrapie-negative sheep brainstem homogenates. Five passages post-inoculation, the transcriptional profiles of mock and infected clones were sequenced using Illumina technology. Comparative transcriptional analyses identified twenty-two differentially transcribed genes, most of which were upregulated in poorly permissive microglia. This included genes encoding for selenoprotein P, endolysosomal proteases, and proteins involved in extracellular matrix remodeling. Furthermore, in highly permissive microglia, transforming growth factor β–induced, retinoic acid receptor response 1, and phosphoserine aminotranspherase 1 gene transcripts were upregulated. Gene Set Enrichment Analysis identified proteolysis, translation, and mitosis as the most affected pathways and supported the upregulation trend of several genes encoding for intracellular proteases and ribosomal proteins in poorly permissive microglia. This study identifies new genes potentially involved in scrapie prion propagation, corroborates results from other studies, and extends those results into another cell culture model. [ABSTRACT FROM AUTHOR]

The prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the population remains uncertain, although it has been estimated that 1 in 2000 people in the United Kingdom are positive for abnormal prion protein (PrPTSE) by a recent survey of archived appendix tissues. The prominent lymphotropism of vCJD prions raises the possibility that some surgical procedures may be at risk of iatrogenic vCJD transmission in healthcare facilities. It is therefore vital that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated. A current limitation is the lack of a rapid model permissive to human prions. Here, we developed a prion detection assay based on protein misfolding cyclic amplification (PMCA) technology combined with stainless-steel wire surfaces as carriers of prions (Surf-PMCA). This assay allowed the specific detection of minute quantities (10−8 brain dilution) of either human vCJD or ovine scrapie PrPTSE adsorbed onto a single steel wire, within a two week timeframe. Using Surf-PMCA we evaluated the performance of several reference and commercially available prion-specific decontamination procedures. Surprisingly, we found the efficiency of several marketed reagents to remove human vCJD PrPTSE was lower than expected. Overall, our results demonstrate that Surf-PMCA can be used as a rapid and ultrasensitive assay for the detection of human vCJD PrPTSE adsorbed onto a metallic surface, therefore facilitating the development and validation of decontamination procedures against human prions. [ABSTRACT FROM AUTHOR]