Glycans are not necessary to maintain the pathobiological features of bovine spongiform encephalopathy.

The role of the glycosylation status of PrPC in the conversion to its pathological counterpart and on cross-species transmission of prion strains has been widely discussed. Here, we assessed the effect on strain characteristics of bovine spongiform encephalopathy (BSE) isolates with different transmission histories upon propagation on a model expressing a non-glycosylated human PrPC. Bovine, ovine and porcine-passaged BSE, and variant Creutzfeldt-Jakob disease (vCJD) isolates were used as seeds/inocula in both in vitro and in vivo propagation assays using the non-glycosylated human PrPC-expressing mouse model (TgNN6h). After protein misfolding cyclic amplification (PMCA), all isolates maintained the biochemical characteristics of BSE. On bioassay, all PMCA-propagated BSE prions were readily transmitted to TgNN6h mice, in agreement with our previous in vitro results. TgNN6h mice reproduced the characteristic neuropathological and biochemical hallmarks of BSE, suggesting that the absence of glycans did not alter the pathobiological features of BSE prions. Moreover, back-passage of TgNN6h-adapted BSE prions to BoTg110 mice recovered the full BSE phenotype, confirming that the glycosylation of human PrPC is not essential for the preservation of the human transmission barrier for BSE prions or for the maintenance of BSE strain properties.

Detection of classical BSE prions in asymptomatic cows after inoculation with atypical/Nor98 scrapie.

The emergence of bovine spongiform encephalopathy (BSE) prions from atypical scrapie has been recently observed upon experimental transmission to rodent and swine models. This study aimed to assess whether the inoculation of atypical scrapie could induce BSE-like disease in cattle. Four calves were intracerebrally challenged with atypical scrapie. Animals were euthanized without clinical signs of prion disease and tested negative for PrPSc accumulation by immunohistochemistry and western blotting. However, an emergence of BSE-like prion seeding activity was detected during in vitro propagation of brain samples from the inoculated animals. These findings suggest that atypical scrapie may represent a potential source of BSE infection in cattle.

5-Methylcytosine and 5-Hydroxymethylcytosine in Scrapie-Infected Sheep and Mouse Brain Tissues.

Scrapie is a neurodegenerative disorder belonging to the group of transmissible spongiform encephalopathies or prion diseases, which are caused by an infectious isoform of the innocuous cellular prion protein (PrPC) known as PrPSc. DNA methylation, one of the most studied epigenetic mechanisms, is essential for the proper functioning of the central nervous system. Recent findings point to possible involvement of DNA methylation in the pathogenesis of prion diseases, but there is still a lack of knowledge about the behavior of this epigenetic mechanism in such neurodegenerative disorders. Here, we evaluated by immunohistochemistry the 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) levels in sheep and mouse brain tissues infected with scrapie. Expression analysis of different gene coding for epigenetic regulatory enzymes (DNMT1, DNMT3A, DNMT3B, HDAC1, HDAC2, TET1, and TET2) was also carried out. A decrease in 5mC levels was observed in scrapie-affected sheep and mice compared to healthy animals, whereas 5hmC displayed opposite patterns between the two models, demonstrating a decrease in 5hmC in scrapie-infected sheep and an increase in preclinical mice. 5mC correlated with prion-related lesions in mice and sheep, but 5hmC was associated with prion lesions only in sheep. Differences in the expression changes of epigenetic regulatory genes were found between both disease models, being differentially expressed Dnmt3b, Hdac1, and Tet1 in mice and HDAC2 in sheep. Our results support the evidence that DNA methylation in both forms, 5mC and 5hmC, and its associated epigenetic enzymes, take part in the neurodegenerative course of prion diseases.

The emergence of classical BSE from atypical/Nor98 scrapie.

Atypical/Nor98 scrapie (AS) is a prion disease of small ruminants. Currently there are no efficient measures to control this form of prion disease, and, importantly, the zoonotic potential and the risk that AS might represent for other farmed animal species remains largely unknown. In this study, we investigated the capacity of AS to propagate in bovine PrP transgenic mice. Unexpectedly, the transmission of AS isolates originating from 5 different European countries to bovine PrP mice resulted in the propagation of the classical BSE (c-BSE) agent. Detection of prion seeding activity in vitro by protein misfolding cyclic amplification (PMCA) demonstrated that low levels of the c-BSE agent were present in the original AS isolates. C-BSE prion seeding activity was also detected in brain tissue of ovine PrP mice inoculated with limiting dilutions (endpoint titration) of ovine AS isolates. These results are consistent with the emergence and replication of c-BSE prions during the in vivo propagation of AS isolates in the natural host. These data also indicate that c-BSE prions, a known zonotic agent in humans, can emerge as a dominant prion strain during passage of AS between different species. These findings provide an unprecedented insight into the evolution of mammalian prion strain properties triggered by intra- and interspecies passage. From a public health perspective, the presence of c-BSE in AS isolates suggest that cattle exposure to small ruminant tissues and products could lead to new occurrences of c-BSE.

Epigenetic Changes in Prion and Prion-like Neurodegenerative Diseases: Recent Advances, Potential as Biomarkers, and Future Perspectives.

Prion diseases are transmissible spongiform encephalopathies (TSEs) caused by a conformational conversion of the native cellular prion protein (PrPC) to an abnormal, infectious isoform called PrPSc. Amyotrophic lateral sclerosis, Alzheimer's, Parkinson's, and Huntington's diseases are also known as prion-like diseases because they share common features with prion diseases, including protein misfolding and aggregation, as well as the spread of these misfolded proteins into different brain regions. Increasing evidence proposes the involvement of epigenetic mechanisms, namely DNA methylation, post-translational modifications of histones, and microRNA-mediated post-transcriptional gene regulation in the pathogenesis of prion-like diseases. Little is known about the role of epigenetic modifications in prion diseases, but recent findings also point to a potential regulatory role of epigenetic mechanisms in the pathology of these diseases. This review highlights recent findings on epigenetic modifications in TSEs and prion-like diseases and discusses the potential role of such mechanisms in disease pathology and their use as potential biomarkers.

Neurogranin and Neurofilament Light Chain as Preclinical Biomarkers in Scrapie.

Prion diseases are diagnosed in the symptomatic stage, when the neuronal damage is spread throughout the central nervous system (CNS). The assessment of biological features that allow the detection of asymptomatic cases is needed, and, in this context, scrapie, where pre-symptomatic infected animals can be detected through rectal biopsy, becomes a good study model. Neurogranin (Ng) and neurofilament light chain (NfL) are proteins that reflect synaptic and axonal damage and have been studied as cerebrospinal fluid (CSF) biomarkers in different neurodegenerative disorders. In this study, we evaluated Ng and NfL both at the protein and transcript levels in the CNS of preclinical and clinical scrapie-affected sheep compared with healthy controls and assessed their levels in ovine CSF. The correlation between these proteins and the main neuropathological events in prion diseases, PrPSc deposition and spongiosis, was also assessed. The results show a decrease in Ng and NfL at the protein and gene expression levels as the disease progresses, and significant changes between the control and preclinical animals. On the contrary, the CSF levels of NfL increased throughout the progression of the disease. Negative correlations between neuropathological markers of prion disease and the concentration of the studied proteins were also found. Although further research is needed, these results suggest that Ng and NfL could act as biomarkers for neurodegeneration onset and intensity in preclinical cases of scrapie.

Distinctive Toll-like Receptors Gene Expression and Glial Response in Different Brain Regions of Natural Scrapie.

Prion diseases are chronic and fatal neurodegenerative diseases characterized by the accumulation of disease-specific prion protein (PrPSc), spongiform changes, neuronal loss, and gliosis. Growing evidence shows that the neuroinflammatory response is a key component of prion diseases and contributes to neurodegeneration. Toll-like receptors (TLRs) have been proposed as important mediators of innate immune responses triggered in the central nervous system in other human neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. However, little is known about the role of TLRs in prion diseases, and their involvement in the neuropathology of natural scrapie has not been studied. We assessed the gene expression of ovine TLRs in four anatomically distinct brain regions in natural scrapie-infected sheep and evaluated the possible correlations between gene expression and the pathological hallmarks of prion disease. We observed significant changes in TLR expression in scrapie-infected sheep that correlate with the degree of spongiosis, PrPSc deposition, and gliosis in each of the regions studied. Remarkably, TLR4 was the only gene upregulated in all regions, regardless of the severity of neuropathology. In the hippocampus, we observed milder neuropathology associated with a distinct TLR gene expression profile and the presence of a peculiar microglial morphology, called rod microglia, described here for the first time in the brain of scrapie-infected sheep. The concurrence of these features suggests partial neuroprotection of the hippocampus. Finally, a comparison of the findings in naturallyinfected sheep versus an ovinized mouse model (tg338 mice) revealed distinct patterns of TLRgene expression.

Prion-Associated Neurodegeneration Causes Both Endoplasmic Reticulum Stress and Proteasome Impairment in a Murine Model of Spontaneous Disease.

Prion diseases are a group of neurodegenerative disorders that can be spontaneous, familial or acquired by infection. The conversion of the prion protein PrPC to its abnormal and misfolded isoform PrPSc is the main event in the pathogenesis of prion diseases of all origins. In spontaneous prion diseases, the mechanisms that trigger the formation of PrPSc in the central nervous system remain unknown. Several reports have demonstrated that the accumulation of PrPSc can induce endoplasmic reticulum (ER) stress and proteasome impairment from the early stages of the prion disease. Both mechanisms lead to an increment of PrP aggregates in the secretory pathway, which could explain the pathogenesis of spontaneous prion diseases. Here, we investigate the role of ER stress and proteasome impairment during prion disorders in a murine model of spontaneous prion disease (TgVole) co-expressing the UbG76V-GFP reporter, which allows measuring the proteasome activity in vivo. Spontaneously prion-affected mice showed a significantly higher accumulation of the PKR-like ER kinase (PERK), the ER chaperone binding immunoglobulin protein (BiP/Grp78), the ER protein disulfide isomerase (PDI) and the UbG76V-GFP reporter than age-matched controls in certain brain areas. The upregulation of PERK, BiP, PDI and ubiquitin was detected from the preclinical stage of the disease, indicating that ER stress and proteasome impairment begin at early stages of the spontaneous disease. Strong correlations were found between the deposition of these markers and neuropathological markers of prion disease in both preclinical and clinical mice. Our results suggest that both ER stress and proteasome impairment occur during the pathogenesis of spontaneous prion diseases.

Evidence of p75 Neurotrophin Receptor Involvement in the Central Nervous System Pathogenesis of Classical Scrapie in Sheep and a Transgenic Mouse Model.

Neurotrophins constitute a group of growth factor that exerts important functions in the nervous system of vertebrates. They act through two classes of transmembrane receptors: tyrosine-kinase receptors and the p75 neurotrophin receptor (p75NTR). The activation of p75NTR can favor cell survival or apoptosis depending on diverse factors. Several studies evidenced a link between p75NTR and the pathogenesis of prion diseases. In this study, we investigated the distribution of several neurotrophins and their receptors, including p75NTR, in the brain of naturally scrapie-affected sheep and experimentally infected ovinized transgenic mice and its correlation with other markers of prion disease. No evident changes in infected mice or sheep were observed regarding neurotrophins and their receptors except for the immunohistochemistry against p75NTR. Infected mice showed higher abundance of p75NTR immunostained cells than their non-infected counterparts. The astrocytic labeling correlated with other neuropathological alterations of prion disease. Confocal microscopy demonstrated the co-localization of p75NTR and the astrocytic marker GFAP, suggesting an involvement of astrocytes in p75NTR-mediated neurodegeneration. In contrast, p75NTR staining in sheep lacked astrocytic labeling. However, digital image analyses revealed increased labeling intensities in preclinical sheep compared with non-infected and terminal sheep in several brain nuclei. This suggests that this receptor is overexpressed in early stages of prion-related neurodegeneration in sheep. Our results confirm a role of p75NTR in the pathogenesis of classical ovine scrapie in both the natural host and in an experimental transgenic mouse model.

Cerebrospinal Fluid and Plasma Small Extracellular Vesicles and miRNAs as Biomarkers for Prion Diseases.

Diagnosis of transmissible spongiform encephalopathies (TSEs), or prion diseases, is based on the detection of proteinase K (PK)-resistant PrPSc in post-mortem tissues as indication of infection and disease. Since PrPSc detection is not considered a reliable method for in vivo diagnosis in most TSEs, it is of crucial importance to identify an alternative source of biomarkers to provide useful alternatives for current diagnostic methodology. Ovine scrapie is the prototype of TSEs and has been known for a long time. Using this natural model of TSE, we investigated the presence of PrPSc in exosomes derived from plasma and cerebrospinal fluid (CSF) by protein misfolding cyclic amplification (PMCA) and the levels of candidate microRNAs (miRNAs) by quantitative PCR (qPCR). Significant scrapie-associated increase was found for miR-21-5p in plasma-derived but not in CSF-derived exosomes. However, miR-342-3p, miR-146a-5p, miR-128-3p and miR-21-5p displayed higher levels in total CSF from scrapie-infected sheep. The analysis of overexpressed miRNAs in this biofluid, together with plasma exosomal miR-21-5p, could help in scrapie diagnosis once the presence of the disease is suspected. In addition, we found the presence of PrPSc in most CSF-derived exosomes from clinically affected sheep, which may facilitate in vivo diagnosis of prion diseases, at least during the clinical stage.

Impairment of autophagy in scrapie-infected transgenic mice at the clinical stage.

Autophagy appears to play a role in the etiology and progress of misfolded protein disorders. Although this process is dysregulated in prion diseases, it is unknown whether this impairment is a cause or a consequence of prion neuropathology. The study of autophagy during the progress of the disease could elucidate its role. For this purpose, we have investigated its regulation at different stages of the disease in Tg338 mice, a transgenic murine model that overexpresses the highly susceptible ovine VRQ prion protein allele. Mice were intracerebrally inoculated with mouse-adapted classical scrapie and euthanized at the preclinical and clinical stages of the disease. Regulation of autophagy was investigated analyzing the distribution of LC3-B and p62 proteins by immunohistochemistry. Moreover, the expression of genes involved in autophagy regulation was quantified by real-time PCR. LC3-B and p62 proteins were downregulated and upregulated, respectively, in the central nervous system of infected mice with clinical signs of scrapie. Accumulation of p62 correlated with scrapie-related lesions, suggesting an impairment of autophagy in highly prion-affected areas. In addition, Gas5 (growth arrest-specific 5), Atg5 (autophagy-related 5), and Fbxw7 (F-box and WD repeat domain containing 7) transcripts were downregulated in mesencephalon and cervical spinal cord of the same group of animals. The impairment of autophagic machinery seems to be part of the pathological process of scrapie, but only during the late stage of prion infection. Similarities between Tg338 mice and the natural ovine disease make them a reliable in vivo model to study prion infection and autophagy side by side.

Forkhead Box P3 Methylation and Expression in Men with Obstructive Sleep Apnea.

BACKGROUND: Epigenetic changes in obstructive sleep apnea (OSA) have been proposed as a mechanism for end-organ vulnerability. In children with OSA, Forkhead Box P3 (FOXP3) DNA methylation were associated with inflammatory biomarkers; however, the methylation pattern and its effect in the expression of this gene have not been tested in adults with OSA. METHODS: Plasma samples from subjects without comorbid conditions other than OSA were analyzed (the Epigenetics Status and Subclinical Atherosclerosis in Obstructive Sleep Apnea (EPIOSA) Study: NCT02131610). In 16 patients with severe OSA (Apnea-Hypopnea Index-AHI- > 30 events/h) and seven matched controls (AHI < 5), methylation of FOXP3 gen was evaluated by PCR of the promoter and by pyrosequencing of the intron 1 Treg-specific demethylated region (TSDR). In another 74 patients with OSA (AHI > 10) and 31 controls, we quantified FOXP3 protein expression by ELISA and gene expression by quantitative real-time PCR. C-reactive protein (CRP) and plasma Treg cells were also evaluated. RESULTS: Neither the levels of the promoter nor the TSDR demethylated region were different between controls and patients with OSA, whether they were grouped by normal or high CRP. FOXP3 protein and mRNA expression did not differ between groups. CONCLUSIONS: FOXP3 methylation or its expression is not altered in adults with OSA, whatever their inflammatory status.

Antimicrobial resistance among canine enteric Escherichia coli isolates and prevalence of attaching-effacing and extraintestinal pathogenic virulence factors in Spain.

The aim of this study was to estimate the prevalence of antimicrobial resistance (AMR) in Escherichia coli from a dog population in Spain and assess specific virulence factors. Susceptibility to 22 antimicrobials was tested along with the production of extended-spectrum ß-lactamases (ESBLs) and AmpC in faecal isolates from 100 dogs. Virulence-related genes associated with attaching and effacing E. coli (eae, Stx1, Stx2) and extraintestinal pathogenic E. coli - ExPEC - (papC, hlyA and cnf1) were detected by PCR. At least one kind of AMR was observed in 73% of the isolates. The highest prevalences corresponded to penicillin (45%), aminoglycoside (40%) and non-extended spectrum cephalosporin (39%) classes. Multidrug resistance (MDR) was observed in 53.4% of the resistant isolates. No resistance to colistin was found. Production of ESBL/AmpC enzymes was detected in 5% of E. coli. Shiga toxin-producing E. coli were not observed, enteropathogenic E. coli were identified in only 12% of them, and ExPEC were found in 25%. Dog faeces can be a source of E. coli strains potentially presenting a threat to humans through their virulence factors or AMR. The non-hygienic keeping of animals may increase the risk of colonisation of such pathogens in humans.

Prion protein polymorphisms associated with reduced CWD susceptibility limit peripheral PrPCWD deposition in orally infected white-tailed deer.

BACKGROUND: Chronic wasting disease (CWD) is a prion disease affecting members of the Cervidae family. PrPC primary structures play a key role in CWD susceptibility resulting in extended incubation periods and regulating the propagation of CWD strains. We analyzed the distribution of abnormal prion protein (PrPCWD) aggregates in brain and peripheral organs from orally inoculated white-tailed deer expressing four different PRNP genotypes: Q95G96/Q95G96 (wt/wt), S96/wt, H95/wt and H95/S96 to determine if there are substantial differences in the deposition pattern of PrPCWD between different PRNP genotypes. RESULTS: Although we detected differences in certain brain areas, globally, the different genotypes showed similar PrPCWD deposition patterns in the brain. However, we found that clinically affected deer expressing H95 PrPC, despite having the longest survival periods, presented less PrPCWD immunoreactivity in particular peripheral organs. In addition, no PrPCWD was detected in skeletal muscle of any of the deer. CONCLUSIONS: Our data suggest that expression of H95-PrPC limits peripheral accumulation of PrPCWD as detected by immunohistochemistry. Conversely, infected S96/wt and wt/wt deer presented with similar PrPCWD peripheral distribution at terminal stage of disease, suggesting that the S96-PrPC allele, although delaying CWD progression, does not completely limit the peripheral accumulation of the infectious agent.

Incidence and characterization of Clostridium difficile in a secondary care hospital in Spain.

INTRODUCTION: Clostridium difficile (C. difficile) is a major nosocomial infectious agent in hospitals. Previous studies have addressed the high proportion of infection episodes that are overlooked in health care facilities. OBJECTIVE: the main aim of this study was to characterize C. difficile clinical cases that occurred in a secondary care hospital during a five-month period. MATERIAL AND METHODS: for this purpose, a total of 137 stool samples from the same number of patients with diarrhea were analyzed for the presence of C. difficile by culture techniques. An enzyme immunoassay (EIA) test for the detection of C. difficile and its toxins was also used in 50 cases (36.5%) for diagnostic purposes. RESULTS: a total of 14 (10.2%) C. difficile isolates were obtained, of which nine (64.3%) were toxigenic. A mean incidence of 3.2 episodes of C. difficile infections (CDI) per 10,000 patients-days was estimated for the study period. Around 56% of the CDI cases were determined as hospital-acquired, whereas 44% originated in the community. Among these, only two episodes (22.2%) were detected in the hospital by the EIA test, which indicated that the hospital CDI detection protocol needed to be revised. One unusual C. difficile isolate was negative for all toxin genes examined and also for the non-toxigenic strain assay, which highlights the need to perform genome sequencing to study its pathogenicity locus insertion site organization. A stable metronidazole-resistant C. difficile strain and three strains showing multidrug resistance were detected in this study, suggesting that C. difficile antimicrobial susceptibility surveillance programs should be established in this health-care facility.

Determining the Relative Susceptibility of Four Prion Protein Genotypes to Atypical Scrapie.

Atypical scrapie is a sheep prion (PrPSc) disease whose epidemiology is consistent with a sporadic origin and is associated with specific polymorphisms of the normal cellular prion protein (PrPC). To determine the relative amounts of PrP polymorphisms present in atypical scrapie, total PrP was digested with chymotrypsin to generate characteristic peptides spanning relevant polymorphisms at positions 136, 141, 154, 171, and 172 of sheep PrPC. A multiple reaction monitoring method (MRM), employing 15N-labeled internal standards, was used to detect and quantify these polymorphisms present in both the PrPSc and PrPC from heterozygous (ALRRY and ALHQY or ALRQD or AFRQY) atypical scrapie-infected or uninfected control sheep. Both polymorphisms of the full length and truncated (C1) natively expressed PrPC are produced in equal amounts. The overall amount of PrPC present in the infected or uninfected animals was similar. PrPSc isolated from heterozygotes was composed of significant amounts of both PrP polymorphisms, including the ALRRY polymorphism which is highly resistant to classical scrapie. Thus, an atypical scrapie infection does not result from an overexpression of sheep PrPC. The replication of all atypical scrapie prions occurs at comparable rates, despite polymorphisms at positions 141, 154, 171, or 172.

Preliminary studies on isolates of Clostridium difficile from dogs and exotic pets.

BACKGROUND: Clostridium difficile infection (CDI) is recognised as an emerging disease in both humans and some animal species. During the past few years, insights into human CDI epidemiology changed and C. difficile is also considered as an emerging community-acquired pathogen. Certain ribotypes (RT) are possibly associated with zoonotic transmission. The objective of this study was to assess the presence of C. difficile in a population of pets and to characterise the isolates. RESULTS: Faecal samples from a total of 90 diarrhoeic dogs and 24 from exotic animal species (both diarrhoeic and non-diarrhoeic) were analysed. Clostridium difficile was isolated from 6 (6.7%) dogs and one reptile sample (4.2%). Four (66.7%) of the six dog strains were capable of producing toxins. Four known different RTs were detected in dogs (010, 014, 123 and 358) and a new one was found in a faecal sample of an exotic animal. This new RT isolate was negative for all toxin genes tested and belonged to sequence type 347 which has been proposed as a Clade-III member. Importantly, two dog strains showed a stable resistance to metronidazole (initial MIC values: 128 and 48 µg/ml). CONCLUSIONS: The results obtained in this study suggest the implementation of antimicrobial susceptibility surveillance programs to assess the prevalence of metronidazole resistance in dogs; molecular studies to elucidate C. difficile metronidazole resistance mechanisms are warranted. Based on the similarity between the ribotypes observed in dogs and those described in humans, the zoonotic transmission should be further explored. Furthermore, exotic animals have shown to harbor uncommon C. difficile strains which require further genomic studies.

Experimental transmission to a calf of an isolate of Spanish classical scrapie.

Multiple theories exist regarding the origin of bovine spongiform encephalopathy (BSE). An early and prominent theory proposed that BSE was the result of the adaptation of sheep scrapie to cattle. The reports to date indicate that the distribution of the pathological prion protein (PrPSc) in experimental bovine scrapie is largely restricted to the central nervous system (CNS). Here, we describe pathological findings in a calf intracerebrally inoculated with a Spanish classical scrapie isolate. While clinical disease was observed 30 months after inoculation and PrPSc was detected in the CNS, the corresponding phenotype differed from that of BSE. Immunohistochemistry and PMCA also revealed the presence of PrPSc in the peripheral nerves, lymphoid tissues, skeletal muscle and gastrointestinal tract, suggesting centrifugal spread of the scrapie agent from the brain. To the best of our knowledge, this is the first report describing the detection of PrPSc in tissues other than the CNS after experimental transmission of scrapie to cattle.

Increased circulating microRNAs miR-342-3p and miR-21-5p in natural sheep prion disease.

Scrapie is a transmissible spongiform encephalopathy (TSE), or prion disease, of sheep and goats. As no simple diagnostic tests are yet available to detect TSEs in vivo, easily accessible biomarkers could facilitate the eradication of scrapie agents from the food chain. To this end, we analysed by quantitative reverse transcription PCR a selected set of candidate microRNAs (miRNAs) from circulating blood plasma of naturally infected, classical scrapie sheep that demonstrated clear scrapie symptoms and pathology. Significant scrapie-associated increase was repeatedly found for miR-342-3p and miR-21-5p. This is the first demonstration, to our knowledge, of circulating miRNA alterations in any animal suffering from TSE. Genome-wide expression studies are warranted to investigate the true depth of miRNA alterations in naturally occurring TSEs, especially in presymptomatic animals, as the presented study demonstrates the potential feasibility of miRNAs as circulating TSE biomarkers.

Quantitating PrP Polymorphisms Present in Prions from Heterozygous Scrapie-Infected Sheep.

Scrapie is a prion (PrPSc) disease of sheep. The incubation period of sheep scrapie is strongly influenced by polymorphisms at positions 136, 154, and 171 of a sheep's normal cellular prion protein (PrPC). Chymotrypsin was used to digest sheep recombinant PrP to identify a set of characteristic peptides [M132LGSXMSRPL141 (X = A or V), Y153XENMY158 (X,= H or R), and Y166RPVDXY172 (X = H, K, Q, or R)] that could be used to detect and quantitate polymorphisms at positions 136, 154, and 171 of sheep PrPC or PrPSc. These peptides were used to develop a multiple reaction monitoring method (MRM) to detect the amounts of a particular polymorphism in a sample of PrPSc isolated from sheep heterozygous for their PrPC proteins. The limit of detection for these peptides was less than 50 attomole. Spinal cord tissue from heterozygous (ARQ/VRQ or ARH/ARQ) scrapie-infected Rasa Aragonesa sheep was analyzed using this MRM method. Both sets of heterozygotes show the presence of both polymorphisms in PrPSc. This was true for samples containing both proteinase K (PK)-sensitive and PK-resistant PrPSc and samples containing only the PK-resistant PrPSc. These results show that heterozygous animals contain PrPSc that is composed of significant amounts of both PrP polymorphisms.