Scrapie In Sheep Research Articles

Modelling of strategies for genetic control of scrapie in sheep: The importance of population structure.

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.

The Structure of PrPSc Prions.

PrPSc (scrapie isoform of the prion protein) prions are the infectious agent behind diseases such as Creutzfeldt–Jakob disease in humans, bovine spongiform encephalopathy in cattle, chronic wasting disease in cervids (deer, elk, moose, and reindeer), as well as goat and sheep scrapie. PrPSc is an alternatively folded variant of the cellular prion protein, PrPC, which is a regular, GPI-anchored protein that is present on the cell surface of neurons and other cell types. While the structure of PrPC is well studied, the structure of PrPSc resisted high-resolution determination due to its general insolubility and propensity to aggregate. Cryo-electron microscopy, X-ray fiber diffraction, and a variety of other approaches defined the structure of PrPSc as a four-rung β-solenoid. A high-resolution structure of PrPSc still remains to be solved, but the four-rung β-solenoid architecture provides a molecular framework for the autocatalytic propagation mechanism that gives rise to the alternative conformation of PrPSc. Here, we summarize the current knowledge regarding the structure of PrPSc and speculate about the molecular conversion mechanisms that leads from PrPC to PrPSc.

Goats singly heterozygous for PRNP S146 or K222 orally inoculated with classical scrapie at birth show no disease at ages well beyond 6 years

Scrapie is a transmissible spongiform encephalopathy of sheep and goats, and scrapie eradication programs in many parts of the world rely on strong genetic resistance to classical scrapie in sheep. However, the utility of putative resistance alleles in goats has been a focus of research because goats can transmit scrapie to sheep and may serve as a scrapie reservoir. Prior work showed that disease-free survival time was significantly extended in orally inoculated goats singly heterozygous for prion amino acid substitutions S146 or K222, but average durations were only around 3 years post-inoculation. The aim of this study was to investigate whether extended survival would exceed 6 years, which represents the productive lifetimes of most commercial goats.While all control homozygotes were clinically affected by an average of <2 years, none of the NS146 or QK222 goats developed clinical scrapie or had PrPSc-positive rectal biopsies. Several NS146 and QK222 goats developed other conditions unrelated to scrapie, but tissue accumulation of PrPSc was not detected in any of these animals. The NS146 heterozygotes have remained disease-free for an average of 2734days (approximately 7.5 years), the longest duration of any classical scrapie challenge experiment with any genotype to date. The QK222 heterozygotes have remained disease-free for an average of 2450days (approximately 6.7 years), the longest reported average duration for QK222 goats challenged with classical scrapie. This research is ongoing, but the current results demonstrate S146 and K222 confer strong resistance to classical scrapie in goats.

Differential effects of divalent cations on elk prion protein fibril formation and stability

ABSTRACTMisfolding of the normally folded prion protein of mammals (PrPC) into infectious fibrils causes a variety of diseases, from scrapie in sheep to chronic wasting disease (CWD) in cervids. The misfolded form of PrPC, termed PrPSc, or in this case PrPCWD, interacts with PrPC to create more PrPCWD. This process is not clearly defined but is affected by the presence and interactions of biotic and abiotic cofactors. These include nucleic acids, lipids, glycosylation, pH, and ionic character. PrPC has been shown to act as a copper-binding protein in vivo, though it also binds to other divalents as well. The significance of this action has not been conclusively elucidated. Previous reports have shown that metal binding sites occur throughout the N-terminal region of PrPC. Other cations like manganese have also been shown to affect PrPC abundance in a transcript-independent fashion. Here, we examined the ability of different divalent cations to influence the stability and in vitro conversion of two variants of PrP from elk (L/M132, 26–234). We find that copper and zinc de-stabilize PrP. We also find that PrP M132 exhibits a greater degree of divalent cation induced destabilization than L132. This supports findings that leucine at position 132 confers resistance to CWD, while M132 is susceptible. However, in vitro conversion of PrP is equally suppressed by either copper or zinc, in both L132 and M132 backgrounds. This report demonstrates the complex importance of ionic character on the PrPC folding pathway selection on the route to PrPSc formation.

Evolutionary biology and the risk of scrapie disease in sheep.

Scrapie in sheep occurs in two forms. Classical scrapie, along with chronic wasting disease in deer, differs from all other prion diseases in being contagious and occurring as propagating epidemics. By contrast, atypical scrapie occurs sporadically and like most other prion diseases, such as Creutzfeldt-Jakob disease in humans, is regarded as non-contagious under natural conditions. This difference motivates the present study, which uses the perspective of evolutionary biology to explore whether atypical or non-contagious scrapie can transform to classical or contagious scrapie when appropriate evolutionary forces are in play. The study begins with an explanation of relevant evolutionary concepts such as evolvability and object of selection and proposes some explicit definitions of key terms such as variation, replication, heritability and strain. Observations of the behaviour of scrapie strains during serial passage in sheep, goats and mice assign the functions of variation, replication and heritability to the scrapie agent and establish it as an object of selection with a capacity for evolvability. Similar observations of strain behaviour in other prion diseases establish prions in general as objects of selection that are able to evolve. Accordingly, it is tenable that non-contagious atypical scrapie can provide progenitors for the prions causing contagious classical scrapie and can be a latent epidemiological threat. A comparison of atypical and classical scrapie identifies an affinity of the scrapie agent for lymphoreticular tissue and predilections for portals of exit and entry as key gain-of-function mutations that endow prion populations in atypical scrapie with the function of communicability or contagiousness. The present study can be seen as the hazard identification step within a process of risk analysis that systematises knowledge in aid of animal health and welfare and biosecurity. It justifies completion of the remaining steps in risk assessment. This task can be assisted by further comparisons of atypical and classical scrapie enlightened by historical records of scrapie disease, the epidemiology of atypical scrapie, the general behaviour of prions and evolutionary biology.

Chapter 23 - Vaccination strategies

Currently all prion diseases are without effective treatment and are universally fatal. It is increasingly being recognized that the pathogenesis of many neurodegenerative diseases, such as Alzheimer disease (AD), includes "prion-like" properties. Hence, any effective therapeutic intervention for prion disease could have significant implications for other neurodegenerative diseases. Conversely, therapies that are effective in AD might also be therapeutically beneficial for prion disease. AD-like prion disease has no effective therapy. However, various vaccine and immunomodulatory approaches have shown great success in animal models of AD, with numerous ongoing clinical trials of these potential immunotherapies. More limited evidence suggests that immunotherapies may be effective in prion models and in naturally occurring prion disease. In particular, experimental data suggest that mucosal vaccination against prions can be effective for protection against orally acquired prion infection. Many prion diseases, including natural sheep scrapie, bovine spongiform encephalopathy, chronic wasting disease, and variant Creutzfeldt-Jakob disease, are thought to be acquired peripherally, mainly by oral exposure. Mucosal vaccination would be most applicable to this form of transmission. In this chapter we review various immunologically based strategies which are under development for prion infection.

The European Union summary report on surveillance for the presence of transmissible spongiform encephalopathies (TSE) in 2016.

This report presents the results of surveillance activities on transmissible spongiform encephalopathies (TSEs) in bovine animals, sheep, goats, cervids and other species, as well as genotyping data in sheep, carried out in 2016 in the European Union according to Regulation (EC) 999/2001, and in Iceland, Norway and Switzerland. In 2016, 1,352,585 bovine animals were tested in the European Union (5% less than in 2015). For the first time, the United Kingdom did not report any case of bovine spongiform encephalopathy (BSE), whereas France reported one classical and three atypical cases (H), and Spain one atypical case (H). The classical BSE case was born after the enforcement of the total EU‐wide feed ban in 2001 (BARB case). In 2016, 286,351 sheep and 110,832 goats were tested (5% and 11% less than in 2015, respectively). Sheep scrapie was reported by 20 Member States (MSs) (685 cases) and goat scrapie by 9 MSs (634 cases). A total of 25 ovine scrapie cases were reported by Iceland and Norway. At the EU level, the occurrence of scrapie in small ruminants remains stable, with classical scrapie (1,175 cases) being reported more frequently than atypical scrapie (135 cases). A total of 97.2% of the classical scrapie cases in sheep occurred in animals with genotypes belonging to the susceptible group, and a random sampling showed that 26.6% of the genotyped sheep held genotypes of the susceptible group (excluding Cyprus). In 2016, five cases of chronic wasting disease were reported in cervids by Norway: three in wild reindeer and two in moose. It was the first time that this disease is reported in Europe. A total of 2,712 cervids were tested for TSEs in seven different member states, 90% of them in Romania, with negative results. A total of 490 animals from other non‐ruminant species were tested in four different member states, with negative results.

Estimating chronic wasting disease susceptibility in cervids using real-time quaking-induced conversion.

In mammals, susceptibility to prion infection is primarily modulated by the host's cellular prion protein (PrPC) sequence. In the sheep scrapie model, a graded scale of susceptibility has been established both in vivo and in vitro based on PrPC amino acids 136, 154 and 171, leading to global breeding programmes to reduce the prevalence of scrapie in sheep. Chronic wasting disease (CWD) resistance in cervids is often characterized as decreased prevalence and/or protracted disease progression in individuals with specific alleles; at present, no PrPC allele conferring absolute resistance in cervids has been identified. To model the susceptibility of various naturally occurring and hypothetical cervid PrPC alleles in vitro, we compared the amplification rates and amyloid extension efficiencies of eight distinct CWD isolates in recombinant cervid PrPC substrates using real-time quaking-induced conversion. We hypothesized that the in vitro conversion characteristics of these isolates in cervid substrates would correlate to in vivo susceptibility - permitting susceptibility prediction for the rare alleles found in nature. We also predicted that hypothetical alleles with multiple resistance-associated codons would be more resistant to in vitro conversion than natural alleles with a single resistant codon. Our studies demonstrate that in vitro conversion metrics align with in vivo susceptibility, and that alleles with multiple amino acid substitutions, each influencing resistance independently, do not necessarily contribute additively to conversion resistance. Importantly, we found that the naturally occurring whitetail deer QGAK substrate exhibited the slowest amplification rate among those evaluated, suggesting that further investigation of this allele and its resistance in vivo is warranted.

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.

Correlation of cellular factors and differential scrapie prion permissiveness in ovine microglia

Prion diseases are fatal neurodegenerative disorders by which the native cellular prion protein (PrPC) is misfolded into an accumulating, disease-associated isoform (PrPD). To improve the understanding of prion pathogenesis and develop effective treatments, it is essential to elucidate factors contributing to cellular permissiveness. We previously isolated five clones from an immortalized subline of ovine microglia, two of which had demonstrated differential permissiveness to a natural isolate of sheep scrapie and distinct transcriptomic profiles. To more robustly identify factors contributing to this activity, relative permissiveness, cell proliferation, selected gene transcript level, and matrix metalloproteinase 2 (MMP2) activity were compared amongst all five clones. Differences in cell proliferation were not detected between clones; however, significant correlations were identified between relative permissiveness and genes associated with cell growth (i.e., RARRES1 and PTN), protein degradation (i.e., CTSB and SQSTM1), and heparin binding (i.e., SEPP1). MMP2 activity varied amongst clones, but did not correlate with permissiveness. These associations support the contribution of cell division and protein degradation on the permissiveness of cultured ovine microglia to PrPD.

Selective propagation of mouse-passaged scrapie prions with long incubation period from a mixed prion population using GT1-7 cells.

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.

Codon 141 polymorphisms of the ovine prion protein gene affect the phenotype of classical scrapie transmitted from goats to sheep

BackgroundA study to investigate transmission of classical scrapie via goat milk was carried out in sheep: firstly, lambs were challenged orally with goat scrapie brain homogenate to confirm transmission of scrapie from goats to sheep. In the second study phase, milk from scrapie-infected goats was fed to lambs. Lambs were selected according to their prion protein gene (PRNP) genotype, which was either VRQ/VRQ or ARQ/ARQ, with or without additional polymorphisms at codon 141 (FF141, LF141 or LL141) of the ovine PRNP. This report describes the clinical, pathological and molecular phenotype of goat scrapie in those sheep that progressed to clinical end-stage.ResultsTen sheep (six VRQ/VRQ and four ARQ/ARQ, of which three FF141 and one LL141) challenged with one of two scrapie brain homogenates, and six pairs of sheep (ARQ, of which five LL141 and seven LF141) fed milk from six different goats, developed clinical disease, which was characterised by a pruritic (all VRQ/VRQ and LL141 sheep) or a non-pruritic form (all LF141 and FF141 sheep). Immunohistochemical (IHC) examination revealed that the pattern of intra- and extracellular accumulation of disease-associated prion protein in the brain was also dependent on PRNP polymorphisms at codon 141, which was similar in VRQ and LL141 sheep but different from LF141 and FF141 sheep. The influence of codon 141 was also seen in discriminatory Western blot (WB), with LF141 and FF141 sheep showing a bovine spongiform encephalopathy-like profile (diminished reactivity with P4 antibody) on brain tissue. However, discriminatory WB in lymphoid tissues, and IHC pattern and profile both in lymphoid and brain tissue was consistent with classical scrapie in all sheep.ConclusionsThis study provided further evidence that the clinical presentation and the pathological and molecular phenotypes of scrapie in sheep are influenced by PRNP polymorphisms, particularly at codon 141. Differences in the truncation of disease-associated prion protein between LL141 sheep and those carrying the F141 allele may be responsible for these observations.

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.

Divergent prion strain evolution driven by PrPC expression level in transgenic mice

Prions induce a fatal neurodegenerative disease in infected host brain based on the refolding and aggregation of the host-encoded prion protein PrPC into PrPSc. Structurally distinct PrPSc conformers can give rise to multiple prion strains. Constrained interactions between PrPC and different PrPSc strains can in turn lead to certain PrPSc (sub)populations being selected for cross-species transmission, or even produce mutation-like events. By contrast, prion strains are generally conserved when transmitted within the same species, or to transgenic mice expressing homologous PrPC. Here, we compare the strain properties of a representative sheep scrapie isolate transmitted to a panel of transgenic mouse lines expressing varying levels of homologous PrPC. While breeding true in mice expressing PrPC at near physiological levels, scrapie prions evolve consistently towards different strain components in mice beyond a certain threshold of PrPC overexpression. Our results support the view that PrPC gene dosage can influence prion evolution on homotypic transmission.

Scrapie, CWD, and Transmissible Mink Encephalopathy.

Transmissible spongiform encephalopathies (TSEs), or prions, are neurodegenerative diseases that affect a variety of animal species, including humans. Cruetzfeldt-Jakob disease (CJD) in humans, sheep and goat scrapie, chronic wasting disease (CWD) of cervids, and transmissible mink encephalopathy (TME) of mink are classified as TSEs. According to the "protein-only" hypothesis (Prusiner, 1982),1 prions are devoid of nucleic acids and consist of assemblies of misfolded host-encoded normal protein, the prion protein (PrPC). Prion propagation is thought to occur by a templating mechanism during which PrPC is recruited, converted to a disease-associated isoform (PrPD), and assembled onto the growing amyloid fibril. This fibular assembly is infectious, with ability to initiate disease processes similar to other pathogenic agents. Evidence indicates that scrapie, CWD, and TME disease processes follow this rule.

The Structure of the Infectious Prion Protein and Its Propagation.

The prion diseases, which include Creutzfeldt-Jakob disease in humans, chronic wasting disease in cervids (i.e., deer, elk, moose, and reindeer), bovine spongiform encephalopathy in cattle, as well as sheep and goat scrapie, are caused by the conversion of the cellular prion protein (PrPC) into a disease-causing conformer (PrPSc). PrPC is a regular, GPI-anchored protein that is expressed on the cell surface of neurons and many other cell types. The structure of PrPC is well studied, based on analyses of recombinant PrP, which is thought to mimic the structure of native PrPC. The mature protein contains an N-terminal, unfolded domain and a C-terminal, globular domain that consists of three α-helices and only a small, two-stranded β-sheet. In contrast, PrPSc was found to contain predominantly β-structure and to aggregate into a variety of quaternary structures, such as oligomers, amorphous aggregates, amyloid fibrils, and two-dimensional crystals. The tendency of PrPSc to aggregate into these diverse forms is also responsible for our incomplete knowledge about its molecular structure. Nevertheless, the repeating nature of the more regular PrPSc aggregates has provided informative insights into the structure of the infectious conformer, albeit at limited resolution. These data established a four-rung β-solenoid architecture as the main element of its structure. Moreover, the four-rung β-solenoid architecture provides a molecular framework for an autocatalytic propagation mechanism, which could explain the conversion of PrPC into PrPSc.

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.

How will prion disease evolve?

Transmissible Spongiform Encephalopathy (TSE) or prion disease is caused by the infectious agent prion or scrapie prion protein (PrPSC). In animals, prion disease includes scrapie in sheep and goat; mink spongiform encephalopathy in mink; chronic wasting disease in elk and deer, and bovine spongiform encephalopathy (BSE), a.k.a. mad cow disease in cattle. Human TSE includes, Kuru; Creutzfeldt-Jakob Disease (CJD); Gerstmann-Straussler-Scheinker Disease (GSS) and more recently, variant CJD, which is thought to be caused by the transmission of BSE to human. The search for the transmissible agent of TSE went through many decades of uncertainty. In contrast to all other known pathogens, the TSE agent was shown to lack nucleic acids and appeared to co-purify with proteins. It was not until the 1980s that the infectious agent of TSE was shown to be a protein. Remarkably, the gene encoding for this protein is present in normal mammals. Subsequently, the word “prion”, an acronym for “proteinases infectious particle” was coined to explain this conundrum. It was postulated that the central event in the pathogenesis of prion diseases is the conversion of the normal cellular prion protein (PrPC) into an intermediate isoform (PrP*) and finally the pathological, protease-resistant and infectious, PrPSC. While the concept of “protein only hypothesis” is firmly established, the processes by which the conversion occurs, and the mechanisms by which PrPSC causes pathogenesis remain incompletely understood. Furthermore, despite exhaustive investigations, the normal physiologic functions of PrPC remain elusive. It is clear, however, that expression of PrPC in the central nervous system (CNS) is required for the neurotoxicity of PrPSC as prnp null mice do not develop neurodegeneration. As a rare neurodegeneration disease, prion disease occurs with 1–3 cases in a million people per year, among which approximately 85% is sporadic without known agents, 10%–15% is genetic due to somatic mutations of the PRNP gene, and less than 1% is infectious. It is likely that the first occurrence of mad cow disease in England was caused by feeding meat and bone meal (MBM) contaminated by prions. How the first prion strain causing scrapie, chronic wasting disease, and transmissible mink encephalopathy remains obscure. For the past years, an international ban on MBM feeding has greatly reduced the occurrence of prion diseases, however, several reasons indicate that prion diseases will not be eradicated. First, somatic mutations of PRNP naturally occur, albeit at a rare frequency. Second, prions can adapt to new hosts, thus increasing their host range and overcoming the transmission barrier. Third, prions remaining in the field are infectious, and in some cases may become more infectious after binding to soil, as evidenced by the spread of chronic wasting disease among otherwise healthy deer populations. Fourth, samples from asymptomatic CJD patients or patients who are misdiagnosed under certain situations can contaminate medical instruments or be transplanted unintentionally, thus causing prion disease. More recently, it is postulated that the prion phenomenon, as defined by “the conversion of a normal protein to become an infectious protein” may also contribute to other neurodegenerative diseases. Hence, studying the pathogenesis of prion disease may provide novel insights into the underlying mechanisms of more common neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s diseases.

Fine morphology and phylogeny of Spiroplasma sp. isolated from eyes of scrapie sheep

Spiroplasma sp. isolated from eyes of sheep with terminal scrapie were reported previously to produce intracellular pathology typical for neurodegenerative diseases in experimentally infected cell lines (Bastian et al., 2014). These isolates have not been yet characterized morphologically or genetically. The paper presents light and electron microscopic description, as well as 16SrDNA sequence data-based phylogenetic analysis of these isolates grown cell-free. The microorganisms were elongated cells of transient helicity up to 10 μm long, and about 100 nm in diameter. Transmission and scanning electron microscopies revealed variable morphology, including presence of helical and non-helical forms. The 16SrDNA from the novel isolates revealed >99.9% similarity to the orthologues from S. chrysopicola and S. syrphidicola, common commensals of tabanid and syrphid flies. On phylogenetic trees the scrapie sheep isolates fell into the S. chrysopicola—S. syrphidicola—TAAS group of Diptera-associated species suggesting likely route of infection through flies contacting eyes of sheep in pastures. The question, whether microorganisms occasionally contaminate eyes of sheep with terminal scrapie upon contacts with flies, or spiroplasmas are linked with the disease, and tabanids play a role of their obligate or facultative vectors and reservoirs, warrants further investigation.

The Structure of Mammalian Prions and Their Aggregates.

Prion diseases, such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle, chronic wasting disease in cervids (i.e., deer, elk, moose, and reindeer), and sheep scrapie, are caused by the misfolding of the cellular prion protein (PrPC) into a disease-causing conformer (PrPSc). PrPC is a normal, GPI-anchored protein that is expressed on the surface of neurons and other cell types. The structure of PrPC is well understood, based on studies of recombinant PrP, which closely mimics the structure of native PrPC. In contrast, PrPSc is prone to aggregate into a variety of quaternary structures, such as oligomers, amorphous aggregates, and amyloid fibrils. The propensity of PrPSc to assemble into these diverse forms of aggregates is also responsible for our limited knowledge about its structure. Then again, the repeating nature of certain regular PrPSc aggregates has allowed (lower resolution) insights into the structure of the infectious conformer, establishing a four-rung β-solenoid structure as a key element of its architecture.