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    Schematic illustration of the algorithm used to determine whether individual sheep and goats require official unique identification as outlined in the NSEP. The requirement for official identification is primarily dependent on the scrapie status of the animal or flock of origin. For animals without movement restrictions, further criteria can be applied to determine whether official identification is required, but the simplest plan is to always apply official identification (ear tag) to an animal that leaves a farm. *Regardless of age, all ewes, rams, does, and bucks that are transported across state lines are required to have an official identification and be accompanied by an interstate certificate of veterinary inspection unless they are being transported directly to a federally approved market or other premises without a change in ownership, in which case they must be accompanied by an owner or hauler statement to that effect. †Includes movement of animals to a terminal feedlot or slaughter-only auction and animals used for personal consumption (ie, home slaughter). ‡Group or lot identification and an owner or hauler statement can be used instead of official individual animal identification for some animal movements. (Adapted from USDA APHIS. Sheep & goat official ID basics flow chart. Available at: www.aphis.usda.gov/animal_health/animal_diseases/scrapie/downloads/sheep-goat-id-flow-chart.pdf. Accessed Feb 12, 2020.)

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Pathogenesis, detection, and control of scrapie in sheep

Eric D. Cassmann DVM, PhD1,2 and Justin J. Greenlee DVM, PhD1
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  • 1 1Virus and Prion Research Unit, National Animal Disease Center, Agricultural Research Service, USDA, Ames, IA 50010.
  • | 2 2Oak Ridge Institute for Science and Education, Oak Ridge, TN 37830.

Abstract

In sheep, scrapie is a fatal neurologic disease that is caused by a misfolded protein called a prion (designated PrPSc). The normal cellular prion protein (PrPC) is encoded by an endogenous gene, PRNP, that is present in high concentrations within the CNS. Although a broad range of functions has been described for PrPC, its entire range of functions has yet to be fully elucidated. Accumulation of PrPSc results in neurodegeneration. The PRNP gene has several naturally occurring polymorphisms, and there is a strong correlation between scrapie susceptibility and PRNP genotype. The cornerstone of scrapie eradication programs is the selection of scrapie-resistant genotypes to eliminate classical scrapie. Transmission of classical scrapie in sheep occurs during the prenatal and periparturient periods when lambs are highly susceptible. Initially, the scrapie agent is disseminated throughout the lymphoid system and into the CNS. Shedding of the scrapie agent occurs before the onset of clinical signs. In contrast to classical scrapie, atypical scrapie is believed to be a spontaneous disease that occurs in isolated instances in older animals within a flock. The agent that causes atypical scrapie is not considered to be naturally transmissible. Transmission of the scrapie agent to species other than sheep, including deer, has been experimentally demonstrated as has the transmission of nonscrapie prion agents to sheep. The purpose of this review is to outline the current methods for diagnosing scrapie in sheep and the techniques used for studying the pathogenesis and host range of the scrapie agent. Also discussed is the US scrapie eradication program including recent updates.

Abstract

In sheep, scrapie is a fatal neurologic disease that is caused by a misfolded protein called a prion (designated PrPSc). The normal cellular prion protein (PrPC) is encoded by an endogenous gene, PRNP, that is present in high concentrations within the CNS. Although a broad range of functions has been described for PrPC, its entire range of functions has yet to be fully elucidated. Accumulation of PrPSc results in neurodegeneration. The PRNP gene has several naturally occurring polymorphisms, and there is a strong correlation between scrapie susceptibility and PRNP genotype. The cornerstone of scrapie eradication programs is the selection of scrapie-resistant genotypes to eliminate classical scrapie. Transmission of classical scrapie in sheep occurs during the prenatal and periparturient periods when lambs are highly susceptible. Initially, the scrapie agent is disseminated throughout the lymphoid system and into the CNS. Shedding of the scrapie agent occurs before the onset of clinical signs. In contrast to classical scrapie, atypical scrapie is believed to be a spontaneous disease that occurs in isolated instances in older animals within a flock. The agent that causes atypical scrapie is not considered to be naturally transmissible. Transmission of the scrapie agent to species other than sheep, including deer, has been experimentally demonstrated as has the transmission of nonscrapie prion agents to sheep. The purpose of this review is to outline the current methods for diagnosing scrapie in sheep and the techniques used for studying the pathogenesis and host range of the scrapie agent. Also discussed is the US scrapie eradication program including recent updates.

The breadth of scientific literature in the field of prion diseases is overwhelming; however, the preponderance of that literature is published in journals that are typically not readily accessible by practicing veterinarians. In fact, a comprehensive review of the numerous intricacies of scrapie has not been published in a journal readily accessible to veterinary practitioners in quite some time. The purpose of this article is to provide an up-to-date review on the pathogenesis, detection, and control of scrapie.

The review begins with the historical context of prion diseases to provide readers with a foundation for understanding the establishment of the protein-only theory. Current understanding of scrapie and other prion diseases presupposes that those diseases are caused by a protein. Next, the functions of PrPC are reviewed along with a discussion of how misfolding of that protein leads to PrPSc accumulation and neurodegeneration. The current state of knowledge surrounding the transmission and pathogenesis of classical scrapie and the genetic determinants that control susceptibility of sheep to classical scrapie are described, including recent evidence regarding new genotypes with a high degree of resistance to the scrapie agent. This is especially important because, historically, eradication of classical scrapie has been largely dependent on selective breeding of animals with resistant genotypes.

Many different strains of scrapie have been described with unique incubation periods and molecular profiles on western blot analysis. Although an indepth review of the theories surrounding prion strain genesis is beyond the scope of this article, we intend to discuss different scrapie strains within a practical and clinical context. Additionally, it is important to understand the differences between classical and atypical scrapie. These 2 variants of the disease have unique characteristics that require different approaches to disease management. Consequently, we have dedicated a separate section in this article to the features of atypical scrapie with an emphasis on highlighting its disease phenotype, transmission, and management characteristics.

It is not unusual for US veterinary professionals to be faced with difficult questions from producers, clients, and the public regarding the diagnosis and epidemiology of prion diseases such as scrapie and CWD. This review addresses current knowledge regarding scrapie including updates on interspecies transmission (ie, the host range) of the scrapie agent and experimental attempts to transmit nonscrapie prion agents to sheep.

The primary goal of the NSEP is to eradicate classical scrapie from the US sheep and goat populations. The program has had great success to date, and this review outlines the NSEP's successes and addresses the challenges it faces moving forward. The USDA APHIS updated regulations in the NSEP final rule on March 25, 2019, and we highlight several of the important changes made.

The History of Prion Diseases and the Protein-Only Theory

Transmissible spongiform encephalopathies are protein-misfolding diseases that lead to fatal neurodegeneration.1 The misfolded protein, denoted PrPSc, is derived from the endogenous cellular prion protein PrPC.2 There are many naturally occurring prion diseases including scrapie in sheep and goats,3 kuru4,5 and Creutzfeldt-Jacob disease6 in humans, BSE in cattle,7,8 CWD in cervids,9,10 feline spongiform encephalopathy in cats,11 transmissible mink encephalopathy in mink,12,13 and the recently described camel prion disease in dromedary camels.14 Scrapie is the oldest recorded prion disease, with references to scrapie dating back at least 300 years.15 In England during the agricultural and textile revolution of the 18th century, scrapie was a politico-economic concern. The disease was even discussed in the British House of Commons in 1775 owing to mounting concerns about its potential adverse economic effects on the merchant trade of woolen products.16 Clinical signs of scrapie are well described in a German article published in 175917; however, the archetypal pathological vacuolation of brain tissue (ie, spongiform encephalopathy) characteristic of scrapie was not described until the end of the 19th century.18

Initial attempts to experimentally transmit scrapie were unsuccessful because insufficient time was allowed for incubation. Successful experimental transmission of scrapie was first documented in the 1930s following observation of sheep for a prolonged period after inoculation with infectious material.19 Despite demonstration of the transmissibility of scrapie, the etiologic agent remained unknown. However, experiments suggested that the etiologic agent was not a typical pathogen because ionizing radiation, extreme heat, and chemicals known to deactivate viruses and bacteria failed to inactivate the causative agent of scrapie.20–22 This was the first step toward a protein-only theory. In 1967, Griffith23 overtly proposed that a protein, and not an organism containing nucleic acid, was the etiologic agent of scrapie. Pursuant to the central dogma of biology, he posed several hypotheses by which a protein could be the etiologic agent of scrapie.23 In one of those hypotheses, he correctly speculated that the etiologic agent of scrapie could be a protein that affected animals were “genetically equipped to make but not in the (deleterious) form.”23 In 1968, Dickinson et al24 crossed F1 progeny mice with a parent, which led to offspring with an autosomal gene without dominance that was linked to the duration of scrapie incubation. The gene was aptly named sinc for scrapie incubation.24 In 1982, 15 years after Griffith first proposed that the etiologic agent of scrapie was a protein, Prusiner1 coined the term prion in what has become the preeminent article on the etiologic agent of scrapie. The most revolutionary concept proposed in that article1 was a protein-only theory that advocated the existence of an infectious protein. Because purification of the scrapie agent had not yet been accomplished, he also proposed that nucleic acid might exist undetected within the core of a tightly packed resilient protein coat.1 Later that year, Prusiner and colleagues isolated and purified scrapie prions.25 The isolated protein was approximately 27,000 to 30,000 Da and consequently was often referred to as PrP27–30 in early studies. PrP27–30 is the main component of the PK-resistant core of PrPSc.26

Hoping to identify viral mRNA in scrapie-infected brains, Chesebro et al27 designed an oligonucleotide probe corresponding to the translated mRNA sequence of PrP27–30. That probe led to the acquisition of a cDNA clone from scrapie-infected brain tissue. The isolated sequence correctly translated back to the amino acid sequence of PrP27–30. Concurrently, the researchers derived the same sequence from brain tissue of clinically normal control mice and hamsters, which suggested that PrP27–30 was most likely derived from an endogenous gene.27 That finding was supported by other contemporaneous research.28 Aside from mice and hamsters, humans were also found to have a gene (PRNP) that encoded PrP27–30.28 PrP27–30 was detected in brain tissue of both scrapie-infected and clinically normal animals, but the protein was not detected in clinically normal brain tissue after application of PK. The protein-only theory was given increasing credibility after susceptibility to scrapie was eliminated in PrP-deficient mice.29 Further convincing evidence was provided by the creation of de novo infectious prions recombinantly expressed in Escherichia coli.30,31 Recombinant de novo prions that exhibited attributes of pathogenic PrPSc were then used to successfully reproduce disease in mice.30,31 Collectively, these data provided overwhelming evidence that prion infectivity and disease are related to conformational alterations in the host's prion protein.

PrPC Function, Structure, and Misfolding

The PrPC is a glycoprotein approximately 231 amino acid residues long32 that is fixed to the plasma membrane of cells via phosphatidylinositol, a glycolipid anchor.33 It includes well-conserved octapeptide repeats that bind cupric (Cu2+) ions in the NH2 terminus.34 In sheep, the highest concentrations of PRNP mRNA transcripts are found in the thalamus and cerebrum followed by the cerebellum, spinal cord, spleen, other lymphoid tissues, brainstem, gastrointestinal tract, and reproductive organs.35

Although many functions of PrPC have been described, its entire range of functions has yet to be elucidated. A prion (PRNP) gene knockout is sublethal in mice, with no obvious phenotypic changes.36 Healthy naturally occurring PrP-deficient goats have been described in Norway.37 Results of experimental studies involving a broad range of testable phenotypic changes indicate that the activity of PrPC is diverse. For example, PrPC-deficient mice have abnormal spatial cognitive abilities that are ameliorated by reintroduction of PrPC to neurons.38 Normal cellular prion protein has a role in synaptic function,39 murine uterine decidualization,40 and sleep regulation.41 Regarding the immune system, PrPC is involved in host-pathogen interactions,42–44 T cell–dendritic cell immunosynapse formation,45 and negative regulation of phagocytosis.46 It also has neuroprotective functions that are related to its binding capabilities.47–52

Prion diseases result from the accumulation of a misfolded form of PrPC, which is designated PrPSc.2 The 2 variants have distinct characteristics evident in their secondary and tertiary structures. The PrPC is comprised of 40% α-helical and 3% β-helical folds, whereas PrPSc is folded into a parallel left-handed β-helical structure that has a 30% α-helical and 40% β-helical conformation.53,54 Individual β sheets organize to form a helical pattern that is purported to associate in a 3- or 4-rung β solenoid that polymerizes into amyloid fibrils.55,56 There are at least 2 proposed models for PrPSc autocatalytic propagation.57 The refolding model assumes that an energy barrier precludes the initial conversion of PrPC to PrPSc; however, transformation of PrPC to PrPSc ensues once that energy barrier is breached or PrPSc is introduced into the system.57 The seeding model asserts that PrPC and PrPSc exist in thermodynamic equilibrium, and PrPSc begins to aggregate when a highly ordered monomeric PrPSc (the seed) stabilizes and recruits more monomeric PrPSc to form larger aggregates. Elucidation of the high-resolution structure of prions and aggregated prions has been challenging because of difficulties associated with the inherent chemical properties of the proteins. However, images of PrPSc rods have been obtained by use of atomic-force and cryoelectron microscopy, which demonstrate that misfolded PrPSc aggregates into repeatable double helical fibers that form 20-nm-wide rods.58 Interestingly, infectious PrPSc-aggregated structures are discernable from non-infectious prion protein fibrils generated in vitro.58

Although aggregation of PrPSc in brain tissue is a pathological feature of prion disease, it is unclear how prion disease results in neurodegeneration. Hypothesized mechanisms by which prion disease triggers neurodegeneration include direct or indirect neurotoxicity, dysregulation of the unfolded protein response, and loss of the normal neuroprotective functions of PrPC. Direct toxic effects of PrPSc on neurons seem unlikely owing to the lack of neuronal lesions in PrPC-depleted mice following inoculation with PrPSc.59,60 However, there is evidence that PrPSc has indirect toxic effects on neurons and causes dysregulation of the cellular unfolded protein response, which induce neurodegenerative changes.61–64 Interestingly, the prototypical vacuolar changes associated with spongiform encephalopathies are not a prerequisite for clinical neurologic signs.65

Determinants of Susceptibility and Resistance to the Scrapie Agent in Sheep

Susceptibility to the classical scrapie agent is determined by the host PrP genotype, exposure age, infectious dose volume, and route of infection. Sheep have several naturally occurring polymorphisms in the PRNP gene. The 3 main codons that have the greatest effect on susceptibility to the scrapie agent are 136, 154, and 171.66–74 Valine at codon 136 (V136), arginine at codon 154 (R154), and glutamine at codon 171 (Q171) are associated with susceptibility to the scrapie agent, especially when they appear together as a V136R154Q171 haplotype. Conversely, the combination of alanine at codon 136 (A136) and arginine at codon 171 (R171) is referred to as the ARR haplotype and is associated with resistance to the scrapie agent. The polymorphic codons 136 and 171 appear to have a greater effect than does codon 154 on the ultimate susceptibility of sheep to scrapie.70,75 Among the common PrP genotypes, resistance to the classical scrapie agent is greatest for the ARR/ARR genotype, followed (in decreasing order) by the ARR/ARQ, ARQ/ARQ, ARR/VRQ, ARQ/VRQ, and VRQ/VRQ genotypes.76 Even though sheep with the ARR/ARR genotype are highly resistant to the scrapie agent, naturally occurring classical scrapie disease has been reported in at least 3 sheep with that genotype.77,78

Substitution of lysine at codon 171 (K171) has been observed in Dorper, Barbados, Barbados–St. Croix crossbred, Suffolk crossbred, and some Mediterranean sheep breeds.79–81 In an epidemiological study80 conducted in Greece, dairy sheep with K171 had a 75% reduction in risk for development of scrapie relative to sheep with Q171. In an experimental study,82 the mean incubation period for sheep with a single K allele at codon 171 (heterozygous QK171; 30 months) was 2.5 times the incubation period sheep that did not have a K allele at codon 171 (homozygous QQ171; 12 months). Results of a more recent study83 indicate that sheep with the A136R154K171 homozygous genotype were resistant to scrapie after oronasal inoculation with PrPSc. In the Greek epidemiological study,80 scrapie was not diagnosed in any sheep with the ARK/ARK or ARR/ARK genotype but was diagnosed in some sheep with the ARQ/ARK genotype. Those findings were congruent with those of a more recent experimental study,83 in which all sheep with the ARQ/ARK genotype developed scrapie following oral inoculation with PrPSc. Results of a similar experimental study74 indicate that sheep with the ARQ/ARR genotype are not susceptible to scrapie following oral inoculation of PrPSc. Thus, it appears that the influence of the K171 polymorphism on resistance to scrapie is greater than that of the Q171 polymorphism but less than that of the R171 polymorphism.

The age at which sheep are first exposed to PrPSc also affects their susceptibility to developing scrapie. Results of statistical modeling that used data from a large flock of Romanov sheep indicate that sheep < 24 months old at the time of initial exposure to PrPSc were 3 times as likely to develop scrapie as were sheep that were initially exposed to PrPSc at ≥ 24 months old.84 Results of other experimental studies85,86 likewise indicate an age-related difference in susceptibility to the scrapie agent. Those studies85,86 involved sheep with the same genotypes and use of the same inoculum and route of inoculation. For lambs that were 12 hours old at the time of PrPSc inoculation,85 the attack rate was greater and the incubation period was shorter, compared with those for lambs that were 4 months old at the time of inoculation and that received 30 times the dose of PrPSc of the 12-hour-old lambs.86 Peyer patches are required for lymphoreticular uptake of PrPSc in the small intestine.87 The age-related effect on susceptibility to scrapie is likely caused by decreasing GALT density because GALT involutes as sheep age. The mean percentage of ileum comprised of Peyer patches ranges from 49.8% to 60.3% in lambs up to 3 months old but is only 0% to 7% in sheep > 18 months old.88 Additionally, FDCs are involved in the pathogenesis of scrapie, and the FDC network within Peyer patches likewise decreases as sheep age.89

Prion Strains

The concept of prion strains refers to the distinct pathological characteristics conferred by different isolates of a TSE agent. Dissimilar strains can result in observable differences in the attack rate, incubation period, clinical signs, extent of brain vacuolation, molecular profile on western blot analysis, susceptible genotypes, and immunolabeling patterns.90–93 A widely used and accepted method for differentiating TSE strains is comparison of the incubation period and extent of vacuolation in brain tissue induced by the respective strains in inbred mice.94–96 However, PrPSc immunolabeling patterns (ie, PrPSc profiling) can be used to differentiate scrapie strains isolated from sheep without passage in mice.92,97,98

Classical Scrapie

Strains

In the United States, different strains of the classical scrapie agent have been identified and isolated from sheep with naturally acquired disease. Field isolates 13–7 and x124 are recognized as separate strains on the basis of differences in their PrPSc immunolabeling profiles, incubation periods, and genotypes of susceptible sheep, as well as the attack rates in C57BL/6 mice.93 Worldwide, strain typing techniques in inbred mice have identified at least 15 distinct prion strains associated with classical scrapie.99 There are also at least 3 distinct PrP western blot profiles documented for classical scrapie isolates from archived samples in the United Kingdom, which are separated into 3 distinct glycoform type categories (A, B, and C) on the basis of their relative mobilities and glycoform ratios observed on western blot analysis.100 Glycoform type A has been isolated from sheep with experimentally induced and naturally acquired scrapie, whereas glycoform type B has been isolated only from sheep with naturally acquired scrapie.100 Differences observed between naturally infected sheep with glycoform type A isolates and those with glycoform type B isolates indicate that there are naturally occurring strains of PrPSc.100 Glycoform type C is an experimental isolate designated CH1641, which was originally isolated from a sheep with naturally acquired scrapie.100 The glycoform profile for CH1641 is similar to C-BSE in sheep.100,101 Naturally occurring CH1641-like strains of the scrapie agent have been identified in sheep throughout Europe.102–104 Initially, there was significant interest in CH1641-like strains because of their biochemical similarities with C-BSE isolates; however, C-BSE and CH1641 strains are distinguishable when experimentally transmitted to mice.102–104 Thus, it is important that biochemical analyses are used in conjunction with other methods to distinguish scrapie strains.

Two mechanisms, the cloud hypothesis and deformed-template hypothesis, have been proposed for the genesis of prion strains associated with classical scrapie. Although the 2 concepts are distinctly defined, they are not mutually exclusive.105 The cloud hypothesis assumes that isolates are comprised of a heterogenous mixture of PrPSc conformations (strains), and over time, a permissive conformer arises to become the predominant variant.106,107 Prevailing conformers are host selected by differences in the replication environment. The deformed-template hypothesis posits that, initially, there is a predominant conformer rather than a mixture of PrPSc conformations, and changes in the replication environment lead to trial-and-error seeding events that generate a new dominant conformer.105 Both hypotheses postulate the existence of multiple conformers (substrains) within an isolate that contribute to the observed differences in disease phenotype.103,108,109 Each conformer or substrain has a distinct capability to replicate in various environmental conditions, and many factors contribute to differences in replication environments. For example, replication environment can be affected by differences in host species or differences in PRNP genotype within a species.

Transmission and pathogenesis in sheep

Classical scrapie is transmitted predominantly via oral consumption of PrPSc by young sheep as evidenced by the detection of the protein in the GALT of lambs exposed to the agent.110–113 Vertical transmission from ewe to lamb occurs prenatally114,115 and during the periparturient period.116,117 Oral transmission of PrPSc during the periparturient period is facilitated by lambs coming into contact with placenta and fetal fluids from infected ewes or environmental items contaminated by those materials.116,117 Nursing lambs can also be orally exposed to PrPSc via colostrum and milk.118,119 Horizontal transmission of scrapie can occur consequent to consumption of PrPSc from a contaminated environment.120 In addition to tissues and fluids associated with the reproductive tract, infected sheep shed PrPSc into the environment through saliva,121,122 urine,123,124 and feces.125 Shedding of PrPSc by infected animals during the subclinical stage of the disease is of particular concern for the control of scrapie.126

The pathogenesis of natural scrapie infection has 3 distinct phases: GALT invasion, lymphatic invasion and dissemination, and neuroinvasion.65,127 After oral exposure, PrPSc crosses the mucosal border of the palatine tonsil, distal portion of the jejunum, and ileum. In the small intestine, PrPSc accumulates in Peyer patches,128 and M cells within Peyer patches are a critical entry point for PrPSc.129 Movement of prions from M cells is facilitated by conventional dendritic cells that express the chemokine receptor CXCR5.130 The presence of prions within Peyer patches is essential for disease development.87 Initial replication of PrPSc occurs in FDCs that express PrPC.131 Results of experimental studies indicate that the susceptibility to scrapie is markedly decreased in FDC-deficient animals; however, FDC-independent pathways for neuroinvasion by PrPSc exist.132–134

During the scrapie incubation period, PrPSc disseminates throughout the lymphoid system.65,128 Afferent drainage of lymph leads to the spread of PrPSc from the palatine tonsil and Peyer patches to the medial retropharyngeal and mesenteric lymph nodes, respectively. It is less clear how PrPSc accumulates in other organs, such as the spleen, that do not receive afferent lymphatic drainage, but hematogenous spread appears likely. The spread of PrPSc by the hematogenous route is supported by the observation of early neuroinvasion in circumventricular organs that lack a blood-brain barrier.135 Also, PrPSc has been detected in the blood of infected sheep.136–139

The ENS is the initial site of neuroinvasion by PrPSc. In an experimental study65 involving sheep, invasion of the ENS by PrPSc did not occur until it had been widely disseminated by the lymphatic system. Neurons within the ENS can become infected with PrPSc by multiple mechanisms. One mechanism is through submucosal fine nerve fibers in close proximity to the mucosal epithelium and lacteals.140 Alternatively, PrPSc may enter the ENS by active cell transport or drainage from Peyer patches.141 This mechanism is supported by the documentation of preferential accumulation of PrPSc in the ENS adjacent to Peyer patches.128,142 After PrPSc invades the ENS, it moves retrograde along efferent axons of parasympathetic and sympathetic neurons to the medulla oblongata and thoracic portion of the spinal cord, respectively.127

In animals with naturally acquired classical scrapie, PrPSc does not stimulate a humoral response because its amino acid sequence is identical to that of PrPC.143,144 Because T cells process antigen as linear epitopes, they cannot distinguish between peptide sequences of PrPSc and PrPC; therefore, antigen-presenting cells are deprived of the necessary T-cell costimulatory signals required to elicit a robust humoral response. Complement components interact with PrPSc, and membrane attack complexes localize in the brain tissue and contribute to the neurodegeneration of scrapie-infected animals.145–147 Additionally, complement-meditated endocytosis may contribute to the movement of PrPSc to lymphoid tissues via dendritic cells expressing CD35 (complement receptor 1).148

It is important to note that most of the current understanding regarding the pathogenesis of classical scrapie is derived from experimental models that involve the use of sheep that are genetically susceptible to the disease. Experimental observation suggests that sheep with genotypes that are less susceptible to scrapie have different propensities for PrPSc invasion of the lymphatic system,149,150 which likely affects the pathogenesis of the disease.

Atypical Scrapie

A novel type of scrapie called Nor98 or atypical scrapie was first described in sheep in Norway in 1998.151 Since then, it has been identified in sheep in the United States.152 Atypical scrapie differs from classical scrapie in that it generally affects single, older animals within a flock; therefore, it is considered a spontaneous prion disease.153 It has also been reported in sheep in Australia154 and New Zealand,155 2 countries that are free of classical scrapie, which provides further evidence of the spontaneity of the disease. Interestingly, atypical scrapie occurs in sheep with genotypes considered to be fairly resistant to classical scrapie.153

The distribution of brain lesions also distinguishes atypical scrapie from classical scrapie. Tissue vacuolation and PrPSc aggregates are found primarily in the cortex of the cerebellum and cerebrum in sheep with atypical scrapie151,156 versus the medulla oblongata, particularly the dorsal motor nucleus of the vagus nerve, in sheep with classical scrapie.

Pathological phenotypic differences between atypical and classical scrapie have been reviewed.157 Unlike sheep with classical scrapie, PrPSc is undetectable by immunoassay in the lymphoid tissues of sheep with atypical scrapie. However, sheep with atypical scrapie were determined to have infectious PrPSc in lymphoid tissue, muscles, and peripheral nervous tissue on the basis of positive bioassay results in transgenic mice that overexpress the sheep prion protein.158 Successful experimental transmission of atypical scrapie to sheep has been achieved with intracranial159 and oral160 inoculation of brain homogenate containing the atypical scrapie agent. However, natural transmission of atypical scrapie among sheep is considered unlikely owing to the low prevalence of the disease across all areas surveyed161 and the fact that only isolated cases of atypical scrapie have been identified; there is no clustering of cases to provide supportive evidence for natural transmission among sheep.162 Consequently, it is believed that transmission of atypical scrapie does not occur or is very inefficient in natural settings.

Methods Used to Diagnose and Research Scrapie

The current standard for identification of PrPSc involves the use of anti-PrP antibodies. Many anti-PrP antibodies are available, and each antibody recognizes a different conformation or linear epitope of PrPSc. Typically, anti-PrP antibodies are generated by injecting PrP-deficient animals with PrPC to induce neutralizing antibodies, which can then be harvested for use in various immunoassays, such as western blot analysis, ELISA, and immunohistochemical, immunocyto-chemical, and immunoprecipitation analyses. Generally, test samples must first undergo digestion with PK to allow for antibody discrimination between PrPC and PrPSc. After controlled PK digestion, PrPC is undetectable, whereas the resistant core of PrPSc (PrP27–30) remains intact for immunodetection.26 Owing to the recognition of unique conformational epitopes, some anti-PrP antibodies are able to selectively bind PrPSc without a sample having to undergo the PK digestion step to eliminate PrPC.163

The preferred postmortem test for diagnosis of scrapie is immunohistochemical analysis for detection of PrPSc in the brainstem at the level of the obex. For most animals with clinical signs of classical scrapie (ie, animals that have neuropathologic changes), PrPSc is widely disseminated and detectable in lymphoid tissues.164 Therefore, immunohistochemical analysis of the lymphoid tissues of the tonsils,165 third eyelid conjunctiva,166 and rectal mucosa167 can be performed to diagnose scrapie in live animals. The acquisition of tonsillar biopsy specimens from live animals requires skill and may require anesthesia. Use of third eyelid conjunctiva for antemortem diagnosis of scrapie has the advantage of being more readily assessible than the tonsils and requires only local analgesia for the acquisition of biopsy specimens. However, only a minimum number of lymphoid follicles are present in approximately 80% of third eyelid biopsy specimens; thus, the estimated diagnostic sensitivity of immunohistochemical staining of third eyelid biopsy specimens for detection of PrPSc ranges from 85% to 90%.168 Other disadvantages associated with the use of third eyelid conjunctiva for antemortem diagnosis of scrapie are that older animals (ie, the animals most likely to develop clinical signs of the disease) tend to have less third eyelid lymphoid tissue and that lymphoid tissue does not regenerate in the third eyelid, which precludes the collection of repeated biopsy specimens at that location.168 When the number of lymphoid follicles per biopsy specimen is considered, rectal mucosa is superior to both tonsil and third eyelid conjunctiva for antemortem diagnosis of scrapie.167 Rectal biopsy specimens can be obtained without anesthesia or sedation, and the rectal mucosa can be biopsied multiple times without a decrease in the number of lymphoid follicles obtained per specimen. At least 10 lymphoid follicles are present in 87% of rectal biopsy specimens, which corresponds to a false-negative test result rate of approximately 9%.167 Additionally, rectal biopsy specimens can be used for immunohistochemical assays in tandem with enzyme immunoassays.169 For any antemortem test for scrapie, negative test results should be re-evaluated multiple times at both the individual and flock level because of the potential effects that PrP genotype, scrapie strain, incubation period, and extent of infection (ie, stage of disease) may have on test performance.

Bioassays involving mice that express transgenic PrPC are useful for evaluating the transmission efficacy of a scrapie agent. For example, Tg338 mice overexpress an ovine VRQ transgene170,171 and are used to analyze sheep scrapie isolates. Transmission of the scrapie agent from a sheep to a mouse expressing ovine PrPC is more efficient than transmission of the agent to a mouse expressing murine PrPC. Furthermore, transgenic mice often overexpress PrPC, which allows prion diseases to be propagated in vivo more rapidly and with greater sensitivity than the disease could be propagated under normal conditions in a natural host over many years. For instance, the classical scrapie strain x124 derived from a sheep with a VRQ/ARR genotype has an incubation time of 9.9 to 13.0 months following intranasal inoculation in sheep with a VRQ/ARQ genotype,93 whereas Tg338 mice administered the same inoculum will succumb to scrapie in approximately 76 days.a The rapid incubation period of prion diseases in transgenic mice allows in vivo experiments to be performed faster and at less expense than similar experiments in the natural hosts.

Experimental models involving mice are widely used to identify prion strains associated with disease. In those models, prion strains are compared on the basis of the extent of vacuolation induced in predefined gray matter and white matter locations.95 Wild-type inbred mice strains C57BL/6, RIII, and VM are regularly used in experimental models of prion diseases; however, the use of inbred mice can be precluded because of species barrier effects and low transmission attack rates. In such cases, the use of transgenic mice that express the host species' PrPC can often result in higher attack rates and shorter incubation periods.171,172 For example, the attack rate for scrapie strain 13–7 was 5.9% (1/17) in C57BL/6 mice on primary passage,93 versus 100% (17/17) in Tg338 mice.a

In vitro assays to identify prions for rapid diagnostic and experimental purposes are also available. The PMCA assay is conceptually analogous to PCR amplification of DNA. For the PMCA method, a sample containing PrPSc is added to brain homogenate substrate from a TSE-free animal, which is then subjected to alternating steps of incubation and sonication.173 Small amounts of undetectable PrPSc are eventually amplified sufficiently to be detectable by western blot analysis or ELISA. More recently, the use of PK-sensitive recombinant PrP in the PMCA assay has decreased the time required to perform the assay from 3 weeks to a couple of days.174 When a sample containing PrPSc is added to brain homogenate containing PK-sensitive recombinant PrP, the undetectable PK-sensitive recombinant PrP is converted to detectable PK-resistant recombinant PrP via sonication.174 The PMCA assay cannot amplify atypical scrapie strains.175 Currently, the PMCA assay is not widely used as a diagnostic tool owing to the potential for spontaneous conversion events that could result in false-positive test results, which could lead to unnecessary restrictions on animal movements or depopulation of flocks.

An alternative to the PMCA assay is the QuIC method, which involves shaking a recombinant protein instead of sonicating a brain homogenate.176 The original QuIC technique identified prions from a sample within a day and relied on laborious western blot analysis. An improved technique called real-time QuIC uses a plate reader and measures the fluorescence of thioflavin T when it interacts with amyloid fibrils.177,178 The intensity of fluorescence increases in response to amyloid formation. With the establishment of a standard curve, this technique can be used to quantify the amount of prions in a sample.179 A more recently described variation of QuIC called endpoint QuIC uses a thermomixer instead of a plate reader to shake samples.180 There are distinct differences in the detection thresholds for prions between conventional real-time QuIC and endpoint QuIC at various time intervals.181 The usefulness of real-time QuIC and ELISA-coupled PMCA assay methods extends beyond simple detection of PrPSc; the real-time QuIC technique has also been used to discriminate prion strains.182,183

The stability of misfolded prions varies among TSE strains, and denaturing PrPSc with increasing concentrations of guanidine hydrochloride allows strains to be segregated into distinct groups.184 Following denaturation with guanidine hydrochloride, the amount of remaining PrPSc is expressed in terms of relative absorbance as determined by ELISA.185,186 This method has been used to distinguish scrapie isolates obtained from US sheep.187

Interspecies Transmission of the Scrapie Agent

Transmission of TSEs among species is often impeded by differences between the host and donor PRNP sequence. Dogs are a good example of a species with a robust barrier to TSE transmission because they are highly resistant to infection with prions and do not appear to be susceptible to TSEs in vivo.188 However, there are documented instances of natural interspecies transmission of TSEs, as evidenced by the transmission of C-BSE to humans,189–191 C-BSE to goats,192 and, likely, L-BSE to mink.193,194 Experimental studies of interspecies TSE transmission are used to assess host ranges and origin of prion strains, and the results of those studies are used to inform policy decisions regarding biosecurity, food security, and the use and disposal of specified risk material.

On the basis of results of experimental studies, cattle appear to have a robust species barrier to the sheep scrapie agent. Cattle were resistant to disease following oral inoculation with scrapie prion strains isolated from US sheep195 as well as scrapie prion strains isolated from sheep in Great Britain during the BSE epizootic in cattle.196 However, cattle did develop disease following intracerebral inoculation of scrapie prion strains isolated from affected sheep in the United States197,198 and Great Britain.199 Interestingly, the disease characteristics of cattle inoculated with the US scrapie strains differed from those of cattle inoculated with the British scrapie strains. Cattle inoculated with the US scrapie stains did not develop spongiform changes, and the primary pathological finding was the aggregation of PrPSc within neurons as determined by immunolabeling,197,198 whereas cattle inoculated with the British scrapie strains developed spongiform changes in the brain.199 Results of additional prion discriminatory methods including western blot analysis and strain typing in transgenic mice and bank voles indicate that the prion strains recovered from cattle inoculated with British scrapie strains were distinct from those associated with C-BSE.199,200 Nonetheless, it is important to note that uncharacterized strains of scrapie could cause BSE-like disease in cattle. Moreover, it cannot be definitively ruled out that the BSE epizootic in Great Britain was not caused by a scrapie prion strain capable of crossing the species barrier of cattle or that was modified during the rendering process and subsequently fed to cattle.

North American white-tailed deer (Odocoileus virginianus) develop disease following intracerebral and oronasal inoculation of the classical scrapie agent.201,202 Deer that were intranasally inoculated with the classical scrapie agent (which simulated a natural route of exposure), accumulated PrPSc with distinct molecular phenotypes on western blot analysis.202 On western blot analysis and depending on the area of the brain examined, the PrPSc recovered from the oronasally inoculated deer had a lower unglycosylated band similar to the scrapie inoculum or a higher unglycosylated band similar to CWD isolates.202

Elk (Cervus canadensis) also develop disease following experimental intracranial inoculation of the sheep scrapie agent.203 The PrPSc immunophenotype recovered from the inoculated elk was similar to that of the scrapie inoculum and distinct from that of isolates obtained from cervids with CWD.203 Those findings suggest that it is unlikely scrapie-affected sheep are the source of CWD in elk.

Several experiments have been conducted in an effort to determine the host range of scrapie. Classical scrapie has been successfully transmitted to raccoons by intracerebral inoculation,204 but raccoons appear to be resistant to atypical scrapie.205 Swine are poorly susceptible to the scrapie agent, and the disease phenotype expressed by pigs following intracerebral inoculation of the scrapie agent is different from that in sheep with scrapie.206 In that study,206 traditional diagnostic methods (western blot analysis, ELISA, and immunohistochemical analysis) failed to detect PrPSc in all 9 pigs assessed 6 months after intracerebral inoculation of the scrapie agent (ie, when they achieved market weight); however, at least 1 of those diagnostic methods yielded positive results for PrPSc in 5 of the 10 pigs evaluated at > 51 months old. Traditional diagnostic methods failed to detect PrPSc in all 24 pigs orally inoculated with the scrapie agent, but results of a mouse bioassay indicated evidence of PrPSc accumulation in some mice following inoculation with homogenated brain substrate from pigs orally inoculated with the scrapie agent.206

The zoonotic potential of the scrapie agent has been investigated. The scrapie agent was originally considered as a possible cause of Creutzfeldt-Jacob disease in humans; however, the incidence of the disease was similar between countries with and without scrapie.207 Results of other research suggest that the zoonotic potential of the scrapie agent is low because t