Viral Properties of Scrapie

ALTHOUGH scrapie agent has many unusual features^1–4, some aspects of its behaviour are analogous to viral properties⁵. The manner in which the agent invades the nervous system after peripheral inoculation immediately recalls such typically neurotropic viruses as poliomyelitis or rabies. After inoculation into the fore-limb changes occur first in the cervical cord; following injection into the leg they appear first in the lumbar cord⁶. We now report another way in which scrapie behaves as a classical virus.     

Environmental Sources of Scrapie Prions

Ovine scrapie and cervine chronic wasting disease show considerable horizontal transmission. Here we report that a scrapie-affected sheep farm has a widespread environmental contamination with prions. Prions were amplified by protein-misfolding cyclic amplification (sPMCA) from seven of nine environmental swab samples taken, including those from metal, plastic, and wooden surfaces. Sheep had been removed from the areas from which the swabs were taken up to 20 days prior to sampling, indicating that prions persist for at least that long. These data implicate inanimate objects as environmental reservoirs for prion infectivity that are likely to contribute to facile disease transmission.Prion diseases are fatal neurological disorders. The archetypal prion disease is scrapie in sheep, and in the last few decades novel prion diseases have emerged in a range of species, including bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer, and variant Creutzfeldt-Jakob disease (vCJD) in humans. The “protein-only” hypothesis dictates that a pathological isoform, PrP^Sc, of the cellular prion protein (PrP^C) constitutes the infectious agent, or prion (13). A wide range of tissues from CWD- and scrapie-affected animals contain PrP^Sc, and affected animals have been shown to excrete or secrete prions in milk, saliva, urine, and feces (2, 3, 6, 7, 8, 10, 16). This finding led to the hypothesis that infectivity resides in the environment, thus explaining the facile transmission of CWD and scrapie. In support of this hypothesis, CWD infectivity has been transmitted from a combination of exposed bedding, water, and food of captive animals (9), and CWD PrP^Sc has been detected within a single environmental water sample (11).Environmental prions are likely to be present at very low levels. The most sensitive method available for the detection of PrP^Sc is serial protein-misfolding cyclic amplification (sPMCA) (1). This technique was pioneered by Soto and colleagues (15), has been used for the amplification of scrapie (17), CWD (4), and vCJD (5) within their natural hosts, and has facilitated the detection of prions within ovine milk (6) and saliva (7) and within cervine urine (4). Here we investigated sources of environmental scrapie prions by applying sPMCA to samples taken from a range of surfaces that were accessible to animals on a farm where scrapie is endemic.Environmental samples were taken from the Veterinary Laboratories Agency, United Kingdom, farm where natural scrapie is endemic, with a high incidence since 1996. Sheep within the flock were exposed to the scrapie agent by natural routes of transmission. Control samples were taken from a farm (ADAS, United Kingdom) that houses a New Zealand-derived scrapie-free flock kept under strict biosecurity conditions. Environmental swabs were taken by wetting foam swabs (Edson Electronics, Northumberland, United Kingdom) in sterile water and then gently swabbing five times in both directions across a surface approximately 10 cm by 2 cm. Two swabs were taken from each area, and all samples were stored at −80^°C. A total of nine environmental samples from a scrapie-affected farm and a scrapie-free farm were analyzed by sPMCA.Two swabs taken from each area were thawed to room temperature and placed in a single container to which 6 ml of 150 mM PO₄ buffer plus 0.5% (vol/vol) Nonidet P-40 and 0.5% (wt/vol) sodium deoxycholate were added. The container was rotated for 2 h. Prions released into this buffer were precipitated on silicon dioxide and then eluted in 200 μl of 0.1% (wt/vol) sodium dodecyl sulfate. Ten microliters of the eluate was amplified within PCR tubes by sPMCA exactly as previously described (7). Samples from both a scrapie-exposed environment and a non-scrapie-exposed environment were analyzed concurrently within the same run on the same sonicator. Each extract was amplified at least in triplicate within a single run and then analyzed by Western blotting (14).Samples from four metal surfaces from an indoor pen occupied by sheep for a few days each week—a gate, a water trough, a feed trough, and penning—were analyzed. Samples from an outdoor environment that had contained sheep 20 days previously—a metal fence, a metal gate, a metal water trough, a plastic post where sheep frequently scratched, and a wooden fence post (Table ​(Table1)—were1)—were also analyzed. After 8 rounds of amplification, PrP^Sc was detected in all samples with the exceptions of the outdoor water trough and gate (Figure ​(Figure11 and Table ​Table1).1). Equivalent samples from a farm housing a scrapie-free flock were also analyzed, and PrP^Sc was not amplified even after 10 rounds of sPMCA. For indoor surfaces from the scrapie-affected farm, 83% of the sPMCA reactions were positive (n = 12), and 0% were positive for equivalent samples from a scrapie-free farm (n = 24). Similarly, 27% of analyses were positive for samples from outdoor surfaces (n = 15), and again no prion was amplified from equivalent samples taken from a scrapie-free farm (n = 30). For comparison of the percentages of positive sPMCA reactions for different cohorts of samples, data were set up as 2 by 2 contingency tables, and Fisher''s exact test (one-tailed) was applied to derive P values. Overall, prions were significantly more likely to be present in the scrapie-affected farm on indoor (P < 0.001) and outdoor (P = 0.009) surfaces. Analyses of all samples were carried out in two independent experiments that gave equivalent results.Open in a separate windowFIG. 1.Amplification of prions from environmental samples. Prions were extracted from swabs taken of surfaces from a scrapie-free farm or a farm where scrapie is endemic. Swabs were taken from a wooden post (1), a plastic scratching post (2), and the following metal surfaces: fencing (3), gate (4 and 6), pen (5), feed trough (7), and water trough (8 and 9). Samples 1, 2, 3, 4, and 8 were taken from outdoor surfaces and 5, 6, 7, and 9 from indoor surfaces. Extracts were used as seeds for 8 rounds of sPMCA. Products were digested with proteinase K and analyzed by Western blotting. PrP was detected with monoclonal antibodies SHA31 and P4. Molecular-weight markers are shown.

TABLE 1.

sPMCA of prions found in the environment^a

Scrapie resistance in ARQ sheep

Variation in the ovine prion protein amino acid sequence influences scrapie progression, with sheep homozygous for A(136)R(154)Q(171) considered susceptible. This study examined the association of survival time of scrapie-exposed ARQ sheep with variation elsewhere in the ovine prion gene. Four single nucleotide polymorphism alleles were associated with prolonged survival. One nonsynonymous allele (T112) was associated with an additional 687 days of survival for scrapie-exposed sheep compared to M112 sheep (odds ratio, 42.5; P = 0.00014). The only two sheep homozygous for T112 (TARQ) did not develop scrapie, suggesting that the allelic effect may be additive. These results provide evidence that TARQ sheep are genetically resistant to development of classical scrapie.     

Isolation of Scrapie Agent from the Placenta of Sheep with Natural Scrapie in Japan

A five-month-pregnant Suffolk sheep histologically diagnosed as spontaneous scrapie was studied. Western blot analysis was performed with rabbit serum against the sheep scrapie-associated fibrils (SAF). In the proteinase K (pk)-treated parental brain and spleen samples, three major bands (15 K, 18 K, and 23 K) were detected. These major bands were not detected from the placenta. Infectious agents were isolated in mice from the brain samples but not from the placental homogenates. In another case of a three-month-pregnant Corriedale sheep without any clinical sign of, but histologically diagnosed as scrapie, was also studied in a similar approach. In the parental brain samples, three major bands (15 K, 18 K and 23 K) were detected. SAF protein was not detected in the parental spleen and placenta. No bands reactive with the antiserum were detected in any other samples from the fetal brain and spleen in both cases. However, infectious agents were isolated in mice from both brain and placental homogenates. Since the placenta is an important site of natural infection, it is worthwhile to study these tissues for the epidemiological study of scrapie infection.     

Scrapie in Sheep in Sweden

In 1986 scrapie was diagnosed in 2 ewes of Swedish landrace (finn sheep) from a herd south of Stockholm (Carlsson et al 1986). As the diagnosis was based on clinical histo-ry and patho-anathomical changes only, inoculation tests in mice and goats were per-formed to try to verify the diagnosis.     

Scrapie I. Transmission and Pathogenesis

Homogenized brain, extracts, and residues of brain from normal and scrapie sheep were inoculated into 116 sheep. Of 72 sheep inoculated with scrapie material 27 developed the disease, whereas four of 44 inoculated with ;normal' brain material showed symptoms similar to those of scrapie. The scrapie agent survived extraction for 18 hours with diethyl ether followed by water for 24 hours. Similarly the agent survived extraction in a Soxhlet apparatus with 95 per cent ethanol containing 2.0 per cent 4 M HC1 for 24 hours. There was no evidence of demyelination in sections of brain and spinal cord supporting the view that allergic encephalomyelitis is not responsible for the scrapie symptoms. Liver function, as measured by the bromsulfalein test, remained normal until just before death. These results preclude that liver dysfunction contributes to the disease but do not exclude the possibilities of other metabolic derangements.     

Endocrinopathy in Mice affected with Scrapie

SCRAPIE is a protracted neurologic disease of sheep that is caused by a virus-like agent¹. The prominent neuropathologic lesions of scrapie are hypertrophy and proliferation of astrocytes, vacuolation of grey matter and progressive degeneration of neurones^2,3. The scrapie agent induces a similar disease in the mouse⁴. Characteristic clinical features include a waddling gait, reluctance to move, dullness, loss of condition and stiffness of the tail. Evidence of endocrinopathy was observed in two inbred strains of mice affected with scrapie. I shall describe pathologic changes in the reproductive organs of the Balb/c mouse and change in coat colour of the C57 black mouse.     

Scrapie prion protein contains a phosphatidylinositol glycolipid

The scrapie (PrPSc) and cellular (PrPC) prion proteins are encoded by the same gene, and their different properties are thought to arise from posttranslational modifications. We have found a phosphatidylinositol glycolipid on both PrPC and PrP 27-30 (derived from PrPSc by limited proteolysis at the amino terminus). Ethanolamine, myo-inositol, phosphate, and stearic acid were identified as glycolipid components of gel-purified PrP 27-30. PrP 27-30 contains 2.8 moles of ethanolamine per mole. Incubation of PrP 27-30 with a bacterial phosphatidylinositol-specific phospholipase C (PIPLC) releases covalently bound stearic acid, and allows PrP 27-30 to react with antiserum specific for the PIPLC-digested glycolipid linked to the carboxyl terminus of the trypanosomal variant surface glycoprotein. PIPLC catalyzes the release of PrPC from cultured mammalian cells into the medium. These observations indicate that PrPC is anchored to the cell surface by the glycolipid.     

Scrapie Pathogenesis in Subclinically Infected B-Cell-Deficient Mice

Prion infections can present without clinical manifestations. B-cell deficiency may be a model for subclinical transmissible spongiform encephalopathy, since it protects mice from disease upon intraperitoneal administration of scrapie prions; however, a proportion of B-cell-deficient mice accumulate protease-resistant prion protein in their brains. Here, we have characterized this subclinical disease. In addition, we have studied the possibility that a neurotoxic factor secreted by B cells may contribute to pathogenesis.     

Absence of Eclipse Phase in Scrapie Mice

Field, Joyce and Keith¹ have claimed that the scrapie agent shows a viral characteristic by having an eclipse phase after being inoculated into the intracerebral region of mice. Three experiments, studying the accumulation of scrapie agent in spleen after intracerebral and intraperitoneal inoculation of mice, have not verified the conclusions of Field et al. A typical curve for the progression of scrapie activity after intracerebral inoculation (Fig. 1) shows no eclipse phase, nor was any observed in an experiment where the titre was examined every 2 days for the first 14 days after inoculation. Moreover, Field and his colleagues have not referred to similar studies with different results from theirs. Four independent groups of workers^2–6 have now examined the levels of scrapie activity in brain and spleen during the early stages after infection by three different routes and in none of these studies was there any clear indication of an eclipse phase in scrapie.     

Simple Method of Evaluating Scrapie in Mice

Mice in advanced scrapie characteristically clasp their hindlegs together. As the disease progresses into the clasping stage, mice exhibit a sequence of identifiable hindleg reactions which have categorized and assigned relative scores permitting objective, quantitative evaluation of disease.     

Scrapie: uncertainties, biology and molecular approaches

The study of the biology of scrapie in sheep is irretrievably associated with the genetics of the PrP gene in sheep. Control of susceptibility and resistance is so closely linked to certain alleles of the sheep PrP gene that no review on scrapie can avoid PrP genetics. Before the importance of PrP protein was discovered and before the influence of the gene itself on disease incidence was understood, it was clear there were some sheep which were more susceptible to natural scrapie than others and that this feature was heritable. These early observations have led to the development and use of PrP genotyping in sheep in what is probably the biggest genetic selection process ever attempted. The accompanying increase in surveillance has also discovered a novel type of scrapie, the subject of much speculation about its origin.     

Scrapie removal using Planova virus removal filters.

As a possible method for reducing the risk of transmissible spongiform encephalopathy (TSE) infection, Planova virus removal filters were tested for their ability to remove scrapie agent ME7. Albumin solution was spiked with high-titre ME7 and filtered through three different pore sizes of Planova filters. Infectivity of the pre- and post-filtration samples was assayed in log dilutions by intracerebral inoculation into C57B1/6 mice. Filtration of albumin solution in the absence or presence of a detergent (Sarkosyl) with Planova 35N (35+/-2 nm mean pore size) removed the contaminating scrapie agent with reduction factors of 4.93 log10 and 1.61 log10, respectively. Filtration, both in the absence and presence of detergent with Planova 15N (15+/-2 nm mean pore size), and in the presence of detergent with Planova 10N (9+/-2 nm mean pore size), showed high levels of scrapie reduction of >5.87 log10, >4.21 log10, and >3.80 log10, respectively, with no residual infectively detected in any of the filtrate samples. The effectiveness of Planova 35N filtration for the removal of infectivity of this TSE agent is greatly reduced in the presence of a strong detergent, but Planova filters with 15 nm or smaller pore size membranes can remove such infectivity at high reduction rates.     

Implications of Conflicting Associations of the Prion Protein (PrP) Gene with Scrapie Susceptibility and Fitness on the Persistence of Scrapie

Background

Existing mathematical models for scrapie dynamics in sheep populations assume that the PrP gene is only associated with scrapie susceptibility and with no other fitness related traits. This assumption contrasts recent findings of PrP gene associations with post-natal lamb survival in scrapie free Scottish Blackface populations. Lambs with scrapie resistant genotypes were found to have significantly lower survival rates than those with susceptible genotypes. The present study aimed to investigate how these conflicting PrP gene associations may affect the dynamic patterns of PrP haplotype frequencies and disease prevalence.

Methodology/Principal Findings

A deterministic mathematical model was developed to explore how the associations between PrP genotype and both scrapie susceptibility and postnatal lamb mortality affect the prevalence of scrapie and the associated change in PrP gene frequencies in a closed flock of sheep. The model incorporates empirical evidence on epidemiological and biological characteristics of scrapie and on mortality rates induced by causes other than scrapie. The model results indicate that unfavorable associations of the scrapie resistant PrP haplotypes with post-natal lamb mortality, if sufficiently strong, can increase scrapie prevalence during an epidemic, and result in scrapie persisting in the population. The range of model parameters, for which such effects were observed, is realistic but relatively narrow.

Conclusions/Significance

The results of the present model suggest that for most parameter combinations an unfavourable association between PrP genotype and post-natal lamb mortality does not greatly alter the dynamics of scrapie and, hence, would not have an adverse impact on a breeding programme. There were, however, a range of scenarios, narrow, but realistic, in which such an unfavourable association resulted in an increased prevalence and in the persistence of infection. Consequently, associations between PrP genotypes and fitness traits should be taken into account when designing future models and breeding programmes.     

The Limits of Test-Based Scrapie Eradication Programs in Goats

Small ruminant post-mortem testing programs were initially designed for monitoring the prevalence of prion disease. They are now considered as a potential alternative to genetic selection for eradicating/controlling classical scrapie at population level. If such policy should be implemented, its success would be crucially dependent on the efficiency of the surveillance system used to identify infected flocks. In this study, we first determined the performance of post-mortem classical scrapie detection in eight naturally affected goat herds (total n = 1961 animals) according to the age at culling. These results provided us with necessary parameters to estimate, through a Monte Carlo simulation model, the performance of scrapie detection in a commercial population. According to this model, whatever the number of tests performed, post mortem surveillance will have limited success in identifying infected herds. These data support the contention that scrapie eradication programs relying solely on post mortem testing in goats will probably fail. Considering the epidemiological and pathological similarities of scrapie in sheep and goats, the efficiency of scrapie surveillance in both species is likely to be similar.