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Ovine Scrapie
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OVINE SCRAPIE New Tools for Control of an Old Disease Katherine 1. O'Rourke, PhD
Ovine scrapie was described as early as 1732, and fascinating accounts of research on this enigmatic disorder have been published in nearly every decade of the 20th century. The transmissible nature of scrapie was demonstrated in 1936, and the resistance of the agent to inactivation by phenol, chloroform, formalin, heat, or irradiation was established within the next 15 years. The occurrence of scrapie in particular bloodlines was convincing enough to warrant consideration of scrapie as a genetic disease for a short time. Subsequent studies demonstrated that scrapie is not an inherited disease; rather, susceptibility to scrapie is under genetic control and typically only sheep of particular genetic backgrounds develop disease in infected flocks. The hallmark microscopic lesion, vacuolated neurons in the medulla oblongata, was described in the late 1800s. Although pathologists continue to debate the diagnostic value of a lesion that occurs at low levels in uninfected sheep, the designation transmissible spongiform encephalopathy (TSE) was applied to scrapie and subsequently to similar neurodegenerative disorders of other mammalian species. Scrapie research intensified in the early 1950s after the importation of infected sheep by Canada, the United States, and Australia. The role of a nucleic acid free agent (the "proteinonly hypothesis") was suggested by several lines of study, and the extensive studies of Stanley Prusiner and his group on an infectious
This work was supported by the Agricultural Research Service, US Department of Agriculture, Grant CWU 5348-32000-015-00D. All material in this article is in the public domain with the exception of any borrowed tables.
From the US Department of Agriculture, Animal Disease Research Unit, Pullman, Washington
VETERINARY CLINICS OF NORTH AMERICA: FOOD ANIMAL PRACTICE VOLUME 17 • NUMBER 2 • JULY 2001
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protein (the prion) culminated in the 1997 Nobel Prize. This was the second such prize to be awarded for work with the TSEs. Carleton Gajdusek's work with kuru, a TSE occurring among a Stone Age tribe in Papua New Guinea, demonstrated that a human TSE could be transmitted experimentally to nonhuman primates. These transmission experiments were suggested by a letter to Dr. Gajdusek from the eminent veterinary neuropathologist William Hadlow, who remarked on the similarities between kuru and ovine scrapie. Kuru was originally thought to be transmitted through cannibalism; subsequent review of the primary data is less exotic and suggests that transmission could occur through inadvertent oral or parenteral exposure to neural tissues during funeral rituals. Oral exposure may be a major route of transmission in natural ovine scrapie after contamination of pastures, barns, feed, or water with a transmissible agent shed in the placenta or fetal fluids of an infected dam. Chronic wasting disease, a TSE of deer and elk, was reported in 1980 and now is endemic in free-ranging cervids in a small area in the western United States and on game farms in several states. Oral exposure to cattle feed contaminated with a heat-resistant transmissible agent probably contributed to epidemic levels of a new bovine spongiform encephalopathy in the United Kingdom, first reported in the late 1980s. Tragically, bovine spongiform encephalopathy is almost certainly the cause of a novel TSE of humans, variant Creutzfeldt-Jakob disease, a disorder currently diagnosed primarily in young adults. The emergence of variant Creutzfeldt-Jakob disease has lent urgency to research on the TSEs and efforts to eradicate scrapie, one of its oldest members. The literature on scrapie and the other TSEs is vast, contentious, and often contradictory. The references in this article are representative of the entire body of work, and the reader is referred to scientific databases for listings of primary research publications. From the clinician's point of view, the most important questions concern transmission, diagnosis, and control of scrapie. Although definitive answers to these questions are not yet known, regulatory agencies and producers are faced with the task of eradicating scrapie while maintaining an economically viable sheep industry in the United States and abroad. This article contains brief descriptions of the TSEs and the proposed causative agent, the associated pathogenesis and pathology, and possible routes of transmission to give clinicians the background for understanding this rapidly changing field of scientific inquiry, the emerging disorders in the TSE family, and new techniques being applied to the diagnosis and control of scrapie. THE FAMILY OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES
Scrapie is the prototype for a diverse group of fatal neurodegenerative disorders classified as TSEs. TSEs of ruminant animals include ovine scrapie, first reported in 173277 and now endemic in many parts of the
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world; bovine spongiform encephalopathy of domestic cattle and captive nondomestic ruminants,95 apparently a food-borne disorder related to the practice of incorporating rendered livestock byproducts into cattle feed; and chronic wasting disease of deer and elk in the United States,9S of unknown etiology but unrelated to bovine spongiform encephalopathy-contaminated feed. TSEs of carnivores include transmissible mink encephalopathy of farm-raised mink,39 of uncertain etiology, and feline spongiform encephalopathy of domestic and nondomestic cats,100 probably originating from exposure to bovine spongiform encephalopathy. The human TSEs include a number of familial disorders associated with point mutations in the PRPN gene. 16 Creutzfeldt-Jakob disease refers to several of these familial diseases, as well as to sporadic TSEs unrelated to PRPN polymorphisms, and to iatrogenic disease associated with corneal transplants, dura mater grafts, or parenteral inoculation with pituitary extracts from inapparently infected donors. Kuru, a human TSE of the Fore tribe of Papua New Guinea, was apparently transmitted by exposure to an infectious agent during handling of tissues during funeral rituals. Variant Creutzfeldt-Jakob disease97 is a newly emerging human TSE associated with exposure to a transmissible agent related to bovine spongiform encephalopathy.13 Variant Creutzfeldt-Jakob disease differs from sporadic, iatrogenic, and familial CreutzfeldtJakob disease in the age distribution of affected individuals, clinical course, diagnostic signs, pathogenic lesion pattern, and distribution of the disease-associated prion protein in extraneural tissues. 56 OVINE SCRAPIE, THE PROTOTYPE TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHY
Most of the reported cases of scrapie in US sheep occur in 3- to 5year-old ewes, although the disease occurs in older ewes, in rams, and in wethers.99 Clinical scrapie occurs at an earlier age (approximately 24 months) in sheep with differing genetic backgrounds in other endemic regions. s,26 Early signs may include behavioral changes (apprehension, failure to stay with the flock, reluctance to approach feed bins) followed by weight loss, incoordination, and loss of wool through rubbing or biting. In some sheep, central nervous system signs become more pronounced over the course of several weeks and include high stepping of the front legs, hopping of the back legs when moving quickly, or inability to rise. Some sheep with prion accumulation in the brain are found dead after stressful events such as shearing, vaccinations, or transport. Transmission
The natural routes of transmission are not known. The incidence of scrapie in lambs born to infected ewes is high and the risk remains high for 60 to 90 days after lambing. 21,44 Unrelated lambs and, to a lesser
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extent, adult sheep and goats may also become infected if housed with infected ewes during lambing. A transmissible agent and the prion protein have been detected in placenta and fetal membranes. 68, 78, 81 Infection may occur through the oral route when infectious tissues or fluids contaminate the bedding, feed, water, or pen surfaces. The agent is probably taken up by lymphoid tissues in the gut/a, 94 disseminated to other lymphoid tissues,35 and eventually spread to the brain through the nervous supply from the nodes. Alternatively, the pathogenic agent may be taken up directly by the enteric nervous system and transported by axonal flow through the vagus nerve to its dorsal motor nucleus,3,40 the site of early prion accumulation in the central nervous system. Causative Agent: The Prion, an Infectious Protein?
The transmissible agent in infectious tissue extracts shows remarkable resistance to inactivation by heat, nucleases, irradiation, and chemical disinfectants (reviewed elsewhere).77,88 The proteinaceous nature of the transmissible agent was suggested by these early inactivation trials. Ownership of the concept of an infectious protein (later termed the prion) is divided among several mathematicians, biochemists, and virologists, but the extensive studies initiated by Stanley Prusiner at the University of California at San Francisco provide the theoretical framework for thousands of additional studies conducted in laboratories around the world. The prion theory79 remains controversial, but no unequivocal demonstration of a conventional microorganism, virus, or subviral particle has yet been made. Prions (PrPs) are expressed as normal cellular sialoglycoproteins, ubiquitously distributed in mammalian tissues. 6, 67 PrP is encoded by the highly conserved PRNP gene,! a single-copy gene with a newly described ancestral copy (PrP doppel).87 The normal cellular isoform is termed PrP-C or PrP-sen to identify it as a normal cellular protein sensitive to digestion with proteases. The abnormal isoform is PrP-Sc (as a generic term for the protein in the disease-associated conformation), or is referred to by disease-specific designations (PrP-Sc, PrP-CJD, PrP-BSE, PrP-CWD; CJD = Creutzfeldt-Jakob disease; BSE = bovine spongiform encephalopathy; CWD = chronic wasting disease) or the biochemical definition PrP-res, to show its relative resistance to proteinases. PrP-Sc is the major component of infectious extracts and may represent the transmissible agent,79 either alone or with yet-undefined cofactors.89 Molecular modeling and in vitro systems7, 17,43,58 suggest that PrPSc molecules, generated endogenously in familial cases or introduced exogenously in transmitted cases, form aggregates with PrP-C. A biochemical trigger or seeding event initiates a structural change in some regions of the PrP-C molecules from the native alpha-helical configuration to the beta sheet folds characteristic of PrP-Sc. This conversion in secondary structure results in characteristic changes in detergent solubility, partial resistance to digestion by some proteinases, and partial resis-
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tance to denaturation by formalin, irradiation, or heat. The remarkably stable PrP-Sc molecules propagated in the cell can then serve as templates for further conversion of PrP-C in other cells or tissues and as the transmissible agent when shed by the infected host. Elegant experiments with gene-ablated PrP-I- "knock-out" mice reconstituted with normal PrP+ I + central nervous system tissue demonstrated that PrP-C is required for PrP-Sc accumulation and disease progression. 10, 14 Furthermore, PrP-C expression in an immune system with functional follicular dendritic cells is required for propagation of extraneural prions and neuroinvasion. s, 9,12 Amplification of the agent in lymphoid tissues is a hallmark of ovine scrapie, and accumulation of prions during the long incubation period is the basis for preclinical diagnosis of scrapie (described in a following section). Pathogenesis
The classic hallmark of scrapie is neuronal loss, although the mechanism of cell death is poorly understood. Accessory cells, including astrocytes and microglia, appear to participate in neuronal 10ss11,30 through release of soluble mediators or excitatory compounds. The direct role of PrP-Sc in pathogenesis remains controversial. Neuronal apoptosis can be induced in neural cultures by exposure to prion protein fragments. 2S, 31, 42 Conversely, accumulation of PrP-Sc in the absence of clinical disease or pathologic changes41 has been reported. The entire spectrum of prion-related molecules and pathogenic mechanisms may take decades to unravel. In the meantime, control of ovine scrapie relies heavily on detection of the PrP-Sc molecule. A better understanding of the molecular pathogenesis of scrapie in natural disease and in experimental models will be critical in the design of drugs needed for intervention in variant Creutzfeldt-Jakob disease and familial TSEs. Diagnosis
Scrapie is a degenerative disease of the central nervous system with no evidence of inflammation or an immune response. Diagnosis of ovine scrapie has traditionally been made after the appearance of suspect clinical signs, with confirmation by light microscopic examination of the brainstem. Histologic diagnosis, however, is based on detection of three nonspecific neuropathologic changes, which may not be apparent during early clinical disease or in autolyzed tissue and may be equivocal even at the clinical phase in some breeds. After development of the prion hypothesis, diagnosis has been supplemented by biochemical detection of PrP-Sc using immunohistochemistry and Western immunoblot analysis. Although examination of the brain remains the gold standard for diagnosing scrapie in sheep with clinical signs, detection of PrP-Sc may be a more accurate approach for diagnosis of preclinically infected
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sheep. The sensitivity of diagnostic testing could be further increased by inclusion of lymphoid tissues of the head and the gut, where PrP-Sc accumulation precedes central nervous system prion propagation by more than a year. Diagnosis by Histology
Scrapie has classically been diagnosed by postmortem examination of the brain for a combination of spongiform change, neuronal degeneration with or without vacuolation, and astrocytosis. 28, 29, 34, 35,74 The extent of spongiform change in the gray matter neuropil varies among breeds of sheep and may be absent or barely detectable. 34 Neurons with large vacuoles in the perikaryon are observed at low levels in normal sheep, and inclusion of this lesion in the diagnostic criteria is based on distribution and extent. 28,101 Astrocytosis, including both hypertrophy and hyperplasia of astrocytes, is a nonspecific reactive response. Although its diagnostic value is debated,28, 29 astrocytosis can be helpful in establishing a histologic diagnosis when the other two signs are minima1. 34 Immunohistochemistry of the Brain
Immunohistochemistry is a useful adjunct to histology because the assay is conducted on sections from the same formalin-fixed, paraffinembedded specimens used for conventional pathology. The earliest accumulation of PrP-Sc in the brain occurs in the dorsal motor nucleus of the vagus nerve, a small structure in the medulla oblongata at the level of the obex. Accurate diagnosis of early cases depends of careful collection of the medulla and proper orientation of the tissue in the paraffin block. There are currently no antibodies that distinguish PrP-Sc from its normal cellular precursor PrP-C in immunohistochemistry; however, formalin fixation, formic acid pretreatment, and mild protease digestion eliminate PrP-C immunoreactivity in many detection systems. 15, 27, 64, 70, 93 Antigen retrieval using high temperatures and either neutral or acid pH buffer increases the sensitivity of PrP-Sc immunohistochemistry on ovine tissues. 37, 38, 65 Immunohistochemistry assays for PrP-Sc using polyclonal rabbit antisera and mouse monoclonal antibodies 69-7l have been described. Polyclonal antisera have the potential advantage of reacting with multiple epitopes on the prion protein but may be lacking in titer, specificity, and quantity. Panels of monoclonal antibodies reacting to distinct sites on the prion protein provide a reasonable compromise between the need for broadly specific reagents for testing sheep of diverse genetic backgrounds and the need for defined specificity and unlimited quantities for large-scale standardization and validation of diagnostic tests. PrP-C co-localizes with PrP-Sc in tissues from infected sheep; the relative amounts of PrP-C and PrP-Sc may vary with breed, age, length of infection, and genotype. The efficacy of pretreatment steps to eliminate the reactivity of PrP-C and the antigen retrieval steps to enhance
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PrP-Sc detection must be validated in sheep of various breeds and genotypes for each combination of primary antibody and detection system. Comparison studies under way in the United States and internationally will result in standardized protocols for detection of PrP-Sc in ruminant tissues. Other Diagnostic Tests for Ruminant Transmissible Spongiform Encephalopathies
If unfixed tissue is available, supplementary diagnostic techniques include Western immunoblot detection of proteinase K-resistant prion protein2, 18,24,62,66,80,82 or detection of scrapie-associated fibrils by electron microscopy.63 Western blot analysis is based on the relative protease resistance of the PrP-Sc protein core. Tissue homogenates from uninfected sheep typically show three bands reactive with anti-PrP antibodies. These bands represent the unglycosylated protein precursor and the successively larger mono- and diglycosylated PrP molecules. Proteinase K digestion hydrolyzes PrP-C into small fragments undetectable under these assay conditions. Proteinase K treatment of PrP-Sc cleaves only an amino terminal fragment, slightly reducing the apparent molecular weight of each of the three bands. Comparison of banding patterns before and after digestion is used to establish a diagnosis. Careful control of the proteinase K digestion step is required to verify that proteinase K digestion is complete despite differences in the amount of tissue in the homogenate, the activity of the proteinase K enzyme lot, or endogenous serine proteinase inhibitors commonly found in some ovine tissues. This type of Western blot test in a kit format (Check Test, Prionics, Zurich, Switzerland)84 is validated for diagnosis of bovine spongiform encephalopathy using brain tissue collected from slaughter cattle in Switzerland and France. In tissues with sparsely distributed PrP-Sc, an enrichment step exploiting the relative insolubility of PrP-Sc in detergent solutions is typically incorporated. 8o,82 Novel test techniques with increased sensitivity or higher throughput are in the development stages. 60, 83, 85 Postmortem Diagnosis of Scrapie' Using Lymphoid Tissues
Accumulation of the transmissible agent in lymphoid tissues precedes deposition in the central nervous system,35 so assay of lymphoid tissues for PrP-Sc is the basis for early diagnostic testing of sheep.55, 80, 92 PrP-Sc was detected by immunohistochemistry assay in a consistently high percentage of lymphoid follicles in 98% (54 of 55) of palatine tonsil samples collected at necropsy from scrapie-infected sheep in the Netherlands.92 Similar observations were made in a smaller study in which tonsil was available from 47 US sheep (31 uninfected and 16 infected animals).69 PrP-Sc was detectable in tonsil tissue of infected sheep at 4 to 8 months of age in flocks with a typical incubation time of 24 months, and at approximately 9 to 14 months in scrapie-infected US sheep with a typical incubation time of 32 to 48 months. The medial
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retropharyngeallymph node is nearly as informative as tonsil, although additional sections must be examined in early cases because PrP-Sc is not distributed uniformly throughout this relatively large lymph node. Additional candidate tissues include the gut-associated lymphoid tissues (ileal Peyer's patches and ileocecal lymph node), presumably the sites of the earliest uptake and propagation of PrP-Sc in orally exposed sheep. In immunohistochemistry studies in the Netherlands and the United States and in an earlier rodent bioassay study,36 PrP-Sc or a transmissible agent was detected in brain but not in tonsil. The value of lymphoidbased tests for early diagnosis will depend in part on an emerging understanding of the pathogenesis of ovine scrapie, including the effects of breed, age, genotype, dose and route of infection, and scrapie strain on distribution of PrP-Sc in peripheral lymphoid tissues. Based on the findings cited, a reliable tissue set for preclinical postmortem diagnosis includes the medulla oblongata at the level of the obex (to include the dorsal motor nucleus of the vagus), tonsil or retropharyngeal lymph node, and ileal Peyer's patches or ileocecal lymph node. Validation of standardized methods is under way in several countries. The US effort includes long-term quarantine of high-risk sheep, with antemortem and postmortem testing by immunohistochemistry and postmortem histology, Western immunoblot, and bioassay of various neural and extraneural tissues in susceptible rodents and ruminants. Antemortem Preclinical Diagnosis by Immunohistochemistry of Peripheral Lymphoid Tissues
Antemortem diagnosis of scrapie was described by Schreuder et al86 using 4-mm samples of tonsillar tissue collected with biopsy forceps using full or partial anesthesia in larger sheep and manual restraint in younger animals. Dr. Steven Parish, a clinician at Washington State University, examined alternative sites of germinal center formation that could be sampled under local anesthesia with disposable instrumentation. Lymphoid tissue on the bulbar surface of the nictitating membrane (the "third eyelid" lymphoid tissue) can be sampled using inexpensive single-use surgical instruments after application of a topical ophthalmic anesthetic.71 This test has been evaluated in clinically affected sheep, in sheep with no exposure to scrapie, and in clinically normal sheep with preclinical disease (Table 1).71 The third-eyelid test is useful in sheep after 14 months of age, assuming that infection is acquired during the perinatal period. Sheep with positive eyelid biopsy samples developed scrapie between 3 and 20 months after testing, when they reached 3 to 4 years of age. The third-eyelid test has completed the first two stages of diagnostic test validation, as described in the Office International des Epizooties Manual of Standards for Diagnostic Tests,57 and has an estimated sensitivity of 88% and a specificity of greater than 97% (Table 1). The test is currently the focus of large-scale validation studies in the United States and Canada.
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Table 1. IMMUNOHISTOCHEMISTRY ASSAY OF THIRD EYELID LYMPHOID TISSUE FOR DIAGNOSIS OF OVINE SCRAPIE Scrapie Status
Number
Eyelid Positive*
Eyelid Negative*
Confirmed scrapie, t clinically suspectt Uninfected sheep§: Sheep with suspect eNS signs (n = 7), clinically normal sheep exposed to scrapie (n = 120), or sheep with no exposure to scrapie (n = 48) Confirmed scrapie: Clinically normal when tested at necropsy (n = 13) or quarantined until progression to clinical disease 3 to 20 months after biopsy (n = 28)
42 175
41 1
1 174
41
36
5
*Results of immunohistochemistry assay performed with MAb F89/160.1.5. tScrapie-infected sheep showed PrP-Sc immunostaining in medulla oblongata at the level of the obex (US samples) or routine histopathology of brain (UK samples). tClinical signs included wool rubbing, weight loss, and ataxia. §Scrapie-uninfected sheep showed no lesions characteristic of scrapie (UK samples) or no PrP-Sc detectable in brain, tonsil, retropharyngeal lymph node, or submandibular lymph node (US samples, n = 127) or had no reported exposure to scrapie in the flock for 5 years (n = 48). Data from O'Rourke KI, Baszler TV, Besser TE, et al: Preclinical diagnosis of scrapie by immunohistochemistry of the third eyelid lymphoid tissue. J Clin Microbio138:3254-3259, 2000.
GENETIC SUSCEPTIBILITY
Scrapie is a transmissible disease in which incubation time and susceptibility to clinical disease are under genetic control. The association between scrapie and particular PRNP alleles has been described for many breeds of sheep under a wide variety of field and experimental conditions. Genetic testing of breeding stock and selection for animals carrying the genes associated with low susceptibility may reduce the risk of scrapie.
The Ovine Prion Gene
The ovine PRNP gene contains the genetic blueprint for the 254 amino acids of the prion protein. One codon (three DNA bases) defines each amino acid. The DNA sequence is variable (polymorphic) at several sites, and each alternative sequence (allele) is named by the corresponding amino acids at these sites, using the single-letter biochemical abbreviation for each amino acid. The codons most closely associated with susceptibility and incubation time are those encoding amino acids 136 (A or V), 154 (R or H), and 171 (Q, R, or H). Predominant alleles for various breeds of sheep in the United Kingdom and the United States are summarized in Table 2.19, 61, 73 Each sheep inherits two copies of the gene, one from each parent. The alleles may be identical (in a homozygous animal) or may differ (in a heterozygous animal), and the combina-
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Table 2. DISTRIBUTION OF PRNP ALLELES REPORTED IN SHEEP IN THE UNITED STATES AND THE UNITED KINGDOM Breeds
Predominant Alleles
ARQ and ARR ARQ, ARR, and VRQ ARQ, AHQ, VRQ, ARR, and ARH ARQ, ARR, and AHQ ARQ, ARR, AHQ, and VRQ
UK Suffolk, UK Cotswold, US Dorset UK Poll Dorset, UK Border Leicester US Suffolk,* UK Texel UK Bluefaced Leicester UK Cheviot, US Cheviot, UK Swaledale, UK Welsh Mountain
*Surveys of US Suffolk sheep71 (unpublished data) show allelic frequencies of approximately 70% ARQ, 25% ARR, and 5% VRQ, AHQ, and ARH. The most common diploid genotypes are ARQ/ ARQ (approximately 45% of surveyed sheep in flock with no selection for scrapie susceptibility), ARQ/ ARR (approximately 35%), ARR/ ARR (approximately 10%), VRQ/ ARQ or VRQ/ ARR (approximately 5%); all other genotypes (approximately 5%).
tion of both alleles is referred to as the animal's scrapie susceptibility
genotype. Association of Scrapie with Ovine Genetics
Forty years ago, Parry75,76 used classical breeding studies in unsuccessful attempts to demonstrate that scrapie is a familial disease. Subsequent studies demonstrated the inheritance of disease susceptibility rather than the disease itself.20, 22, 45, 46, 48 The association between specific alleles of the PRNP gene and clinical scrapie has been demonstrated under a wide variety of field and experimental conditions in several breeds.* Variation at codon 154 modulates incubation time in some breeds and is associated with some reduction in susceptibility in other breeds. 19,90 Susceptibility is more clearly associated with variation at codons 136 and 171 in most breeds examined. In breeds in which the VRQ allele occurs at high frequency, scrapie is found in sheep with one or two VRQ alleles. Sheep lacking this allele resist experimental challenge and are at low risk of scrapie. In breeds generally lacking the VRQ allele, scrapie susceptibility is controlled by polymorphisms at codon 171. Sheep homozygous for 171Q are susceptible to experimental and natural scrapie; sheep with at least one ARR allele rarely develop clinical disease or PrP-Sc accumulation in brain or lymphoid tissues. Potential Drawbacks to Genetic Selection
Based on the numerous studies cited previously, relative genetic susceptibility to scrapie in sheep homozygous or heterozygous for the *References 4, 8, 22, 23, 32, 33, 47, 49-54, 59, 61, 68, 72, 90, 91, and 96.
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VRQ allele or homozygous for the ARQ allele is well accepted. The relative scrapie "resistance" of sheep with the genotypes ARQ/ ARR or ARR/ ARR is less well defined. Scrapie has been reported in a small number of sheep with these genotypes. 54,91 There is also some concern that heterozygous ARQ/ ARR sheep may develop a silent carrier state, shedding the transmissible agent while remaining clinically normal. Data from a large field study in France23 failed to demonstrate such a carrier state in heterozygous ARQ/ ARR sheep, and PrP-Sc has not yet been detected in brain or lymphoid tissues of ARR sheep in studies in this laboratory (unpublished data, 1990-2000). Studies monitoring sheep for a possible carrier state are in progress in the United States, the United Kingdom, and Europe. Scrapie Susceptibility Genetics and Control Programs
The United Kingdom has proposed a scrapie risk reduction program based on increasing the frequency of the ARR allele in purebred stock. Rams are scored for relative susceptibility by diploid genotype, ranging from highly susceptible (RS) through relatively resistant (R1) (Table 3). The relative risk of scrapie in first-generation progeny is weighted more heavily than relative risk to the breeding ram in this scheme. Only rams with the lower susceptibility scores R1 and R2 are to be used in the proposed UK National Scrapie Plan. In the United States, one state (Michigan) has proposed a scrapie control program based on gradual transition to R1 genotypes in breeding stock, and a second state (Washington) limits the movement of rams with the RS genotype. The contribution of scrapie susceptibility genotyping in scrapie eradication is under investigation in a large-scale field trial in the United States. PREVENTION AND CONTROL
The risk of scrapie can be reduced through a coordinated program of flock management, genetics, and preclinical testing. Purchase of healthy animals is the cornerstone of a good management program. In the United States, state and federal programs such as the US Department of Agriculture Voluntary Scrapie Flock Certification Program can provide flock owners with a source of sheep from flocks monitored for scrapie. Purchased ewes from other flocks, particularly ewes of the high-susceptibility genotype, should be considered a potential risk. Good lambing records, including date and month of lambing with information on housing of ewes during this period, should be maintained and kept for 6 years. Provision of separate lambing quarters for genetically susceptible purchased ewes, with eyelid testing at least 14 months after arrival and culling of test-positive ewes, may reduce the risk of transmission to other animals in the flock. Good sanitation, including prompt disposal of placental tissue, fetal membranes, and contaminated bedding, with
POSED SCORING SYSTEM FOR SELECTING BREEDING RAMS BASED ON ESTIMATED RISK OF SCRAPIE IN THE RA -GENERATION PROGENY Risk of Scrapie Infection in Predominant Genotypes
ARR/ARR ARRlAHQ or AHQ/ AHQ ARRlARQ ARRIARH
ARQ/AHQ ARH/ARH ARQ/ARH ARQ/ARQ ARR/VRQ AHQ/VRQ VRQ combined with any other allele
Ram Type in Propos UK National Scrapie P
Ram
First Generation
Very low Low Low
Low Low Variable (dependent on genotype of ewe)
Type I Type II genotypes in bold Type II genotypes in bold
Medium
Generally higher than progeny of R3 rams
Only used during the first 2 yea Type I and Type II rams are not
High
Generally higher than progeny of R3 and R4 rams
Not allowed
sk of scrapie in ram and first-generation progeny from very low (R1) to very high (R5) after natural exposure to scrapie in the flock. I and Type II rams could be used for breeding stock; progeny of Type II rams would require continued genetic testing. m Dawson M, Hoinville LJ, Hosie BD, et al: Guidance on the use of PrP genotyping as an aid to the control of clinical scrapie. Vet Rec 142:623-
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cleaning of implements and washable surfaces with a solution of 40% household bleach88 should reduce the level of the infectious agent in lambing quarters. Eyelid testing of live sheep may be recommended or required by regulatory agencies after possible exposure to scrapie. Postmortem testing of brain, tonsil, and ileocecal lymph node of sheep dying of unknown causes is recommended, particularly for high-susceptibility sheep purchased from flocks of unknown scrapie status. A longterm, cost-effective preventive approach is identification of candidate rams with good breeding potential, followed by codon 171 testing and selection of 171RR (Type I) or 171QR (Type II) rams from that group. Fully susceptible 171QQ rams with superior breeding characteristics can be used with 171RR ewes to increase the supply of rams for this program. Large-scale surveillance of slaughter samples and random live animal testing at fairs and sales may eventually be used to identify infected flocks. An integrated program of management, preclinical testing, and genetics can prevent the introduction and spread of scrapie within flocks and may eventually contribute to the eradication of scrapie in the United States.
VETERINARIANS' ROLES IN SCRAPIE ERADICATION
Scrapie eradication in the United States involves veterinarians as research scientists, pathologists, diagnosticians, and regulatory personneL Most importantly, veterinary clinicians represent the interface between producers and a bewildering array of rapidly changing recommendations, regulations, and diagnostic tests. In the early stages of an eradication program, an increasing number of infected flocks will be identified by these tests, with subsequent losses of sheep that are often known to the producer by name as well as eartag number or sale price. The veterinarian will be charged with educating producers about the need for stringent control programs, but he or she will also serve by relaying to the regulatory bodies the need for compassion in the development and conduct of a scrapie eradication program. Resources for veterinarians can be found through continuing education programs and through the US Department of Agriculture / Agricultural Research Service (Animal Disease Research Unit: http://pwa.ars. usda.gov / adru) or Animal and Plant Health Inspection Service (Veterinary Services Scrapie Control Programs: http:/ / www.aphis.usda. gov /vs/scrapie). Information on the British-proposed National Scrapie Plan based on ram genetics is found at http://www.maff.gov.uk/ animalh/bse /bsescience / scrapie / nsp / nsp.htmL Commercial genotyping laboratories in the United States are Livestock Molecular Research and Development (Monticello, IL; telephone: 217-762-3094) and GeneCheck (Fort Collins, CO; telephone: 800-822-6740).
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References 1. Basler K, Oesch B, Scott M, et al: Scrapie and cellular PrP isoforms are encoded by the same chromosomal gene. Cell 46:417--428, 1986 2. Beekes M, Baldauf E, Cabens 5, et al: Western blot mapping of disease-specific amyloid in various animal species and humans with transmissible spongiform encephalopathies using a high-yield purification method. J Gen Virol 76:2567-2576, 1995 3. Beekes M, McBridePA, Baldauf E: Cerebral targeting indicates vagal spread of infection in hamsters fed with scrapie. J Gen Virol 79:601-607, 1998 4. Belt PBGM, Muileman IH, Schreuder BEe, et al: Identification of five allelic variants of the sheep PrP gene and their association with natural scrapie. J Gen Virol 76:509517, 1995 5. Blattler T, Brandner S, Raeber AJ, et al: PrP-expressing tissue required for transfer of scrapie infectivity from spleen to brain. Nature 389:69-73, 1997 6. Bolton DC, Meyer RK, Prusiner SB: Scrapie PrP 27-30 is a sialoglycoprotein. J Virol 53:596-606, 1985 7. Bossers A, Belt PBGM, Raymond GJ, et al: Scrapie susceptibility-linked polymorphisms modulate the in vitro conversion of sheep prion protein to protease-resistant forms. Proc Natl Acad Sci USA 94:4931--4936, 1997 8. Bossers A, Schreuder BEC, Muileman IH, et al: PrP genotype contributes to determining survival times of sheep with natural scrapie. J Gen Virol 77:2669-2673, 1996 9. Brandner S, Isenmann S, Raeber A, et al: Normal host prion protein necessary for scrapie-induced neurotoxicity. Nature 379:339-343, 1996 10. Brandner S, Raeber A, Sailer A, et al: Normal host prion protein (Prpc) is required for scrapie spread within the central nervous system. Proc Natl Acad Sci USA 93:13148-13151, 1996 11. Brown DR: Prion protein peptide neurotoxicity can be mediated by astrocytes. J Neurochem 73:1105-1113, 1999 12. Brown KL, Stewart K, Ritchie DL, et al: Scrapie replication in lymphoid tissues depends on prion protein-expressing follicular dendritic cells. Nat Med 5:1308-1312, 1999 13. Bruce ME: "New variant" Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Nat Med 6:258-259, 2000 14. Bueler H, Aguzzi A, Sailer A, et al: Mice devoid of PrP are resistant to scrapie. Cell 73:1339-1347, 1993 15. Castellani RJ, Parchi P, Madoff L, et al: Biopsy diagnosis of Creutzfeldt-Jakob disease by Western blot: A case report. Hum Pathol 28:623-626, 1997 16. Collinge J, Palmer MS: Molecular genetics of human prion diseases. Philos Trans R Soc Lond BioI Sci 343:371-378, 1994 17. Come JH, Fraser PE, Lansbury PT: A kinetic model for amyloid formation in the prion diseases: Importance of seeding. Proc Natl Acad Sci USA 90:5959-5963, 1993 18. Cooley WA, Clark JK, Stack MJ: Comparison of scrapie-associated fibril detection and Western immunoblotting for the diagnosis of natural ovine scrapie. J Comp Pathol 118:41--49, 1998 19. Dawson M, Hoinville LJ, Hosie BD, et al: Guidance on the use of PrP genotyping as an aid to the control of clinical scrapie. Vet Rec 142:623-625, 1998 20. Dickinson AG, Meikle VM: Host-genotype and agent effects in scrapie incubation: Change in allelic interaction with different strains of agent. Mol Gen Genet 112:7379, 1971 21. Dickinson AG, Stamp JT, Renwick CC: Maternal and lateral transmission of scrapie in sheep. J Comp Pathol 84:19-25, 1974 22. Dickinson AG, Stamp JT, Renwick CC, et al: Some factors controlling the incidence of scrapie in Cheviot sheep injected with a Cheviot-passaged scrapie agent. J Comp Pathol 78:313-321, 1968 23. Elsen JM, Amigues Y, Schelcher F, et al: Genetic susceptibility and transmission factors in scrapie: Detailed analysis of an epidemic in a closed flock of Romanov. Arch Viro1144:431--445, 1999
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24. Farquhar CF, Somerville RA, Ritchie LA: Post-mortem immunodiagnosis of scrapie and bovine spongiform encephalopathy. J Virol Methods 24:215-222, 1989 25. Forloni G, Angeretti N, Chiesa R, et al: Neurotoxicity of a prion protein fragment. Nature 362:543-546, 1993 26. Foster JD, Dickinson AG: Genetic control of scrapie in Cheviot and Suffolk sheep. Vet Rec 123:159, 1988 27. Foster JD, Wilson M, Hunter N: Immunolocalisation of the prion protein (PrP) in the brains of sheep with scrapie. Vet Rec 139:512-515, 1996 28. Fraser H: The pathology of natural and experimental scrapie. Front BioI 44:267-305, 1976 29. Georgsson G, Gisladottir E, Arnadottir S: Quantitative assessment of the astrocyte response in natural scrapie of sheep. J Comp Pathol108:229-240, 1993 30. Giese A, Brown DR, Groschup MH, et al: Role of microglia in neuronal cell death in prion disease. Brain Pathol 8:449-457, 1998 31. Giese A, Groschup MH, Hess B, et al: Neuronal cell death in scrapie-infected mice is due to apoptosis. Brain Pathol 5:213-221, 1995 32. Goldmann W, Hunter N, Foster JD, et al: Two alleles of a neural protein gene linked to scrapie in sheep. Proc Natl Acad Sci USA 87:2476-2480, 1990 33. Goldmann W, Hunter N, Smith G, et al: PrP genotype and agent effects in scrapie: Change in allelic interaction with different isolates of agent in sheep, a natural host of scrapie. J Gen Virol 75:989-995, 1994 34. Hadlow WJ: Differing neurohistologic images of scrapie, transmissible mink encephalopathy, and chronic wasting disease of mule deer and elk. In Gibbs CJ Jr (ed): Bovine Spongiform Encephalopathy: The BSE Dilemma. New York, Springer, 1996, pp 122-137 35. Hadlow WJ, Kennedy RC, Race RE: Natural infection of suffolk sheep with scrapie virus. J Infect Dis 146:657-664, 1982 36. Hadlow WJ, Rice RE, Kennedy RC, et al: Natural infection of sheep with scrapie virus. In Prusiner SB, Hadlow WJ (eds): Slow Transmissible Diseases of the Nervous System. New York, Academic Press, 1979, pp 3-12 37. Hardt M, Baron T, Groschup MH: A comparative study of immunohistochemical methods for detecting abnormal prion protein with monoclonal and polyclonal antibodies. J Comp Pathol 122:43-53, 2000 38. Haritani M, Spencer YI, Wells GAH: Hydrated autoclave pretreatment enhancement of prion protein immunoreactivity in formalin-fixed-bovine spongiform encephalopathy-affected brain. Acta Neuropathol (Berl) 87:86-90, 1992 39. Hartsough GR, Burger D: Encephalopathy of mink: 1. Epizootiologic and clinical observations. J Infect Dis 115:387-392, 1965 40. Heggebo R, Press CM, Gunnes G, et al: Distribution of prion protein in the ileal Peyer's patch of scrapie-free lambs and lambs naturally and experimentally exposed to the scrapie agent. J Gen Virol 81:2327-2337, 2000 41. Hill AF, Joiner 5, Linehan J, et al: Species-barrier-independent prion replication in apparently resistant species. Proc Natl Acad Sci USA 97:10248-10253, 2000 42. Hope J, Shearman MS, Baxter HC, et al: Cytotoxicity of prion protein peptide (PrP 106-126) differs in mechanism from the cytotoxic activity of the Alzheimer's Disease amyloid peptide, AB25-35. Neurodegeneration 5:1-11, 1996 43. Horiuchi M, Priola SA, Chabry J, et al: Interactions between heterologous forms of prion protein: Binding, inhibition of conversion, and species barriers. Proc Natl Acad Sci USA 97:5836-5841, 2000 44. Hourrigan JL, Klingsporn AL, Clark WW, et al: Epidemiology of scrapie in the US. In Prusiner SB, Hadlow WJ (eds): Slow Transmissible Diseases of the Nervous System. New York, Academic Press, 1979, pp 331-356 45. Hunter N, Cairns D: Scrapie-free Merino and Poll Dorset sheep from Australia and New Zealand have normal frequencies of scrapie-susceptible PrP genotypes. J Gen Virol 79:2079-2082, 1998 46. Hunter N, Cairns D, Foster JD, et al: Is scrapie solely a genetic disease? Nature 386:137, 1997 47. Hunter N, Foster JD, Benson G, et al: Restriction fragment length polymorphisms of
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71. O'Rourke KI, Baszler TV, Parish SM, et al: Preclinical detection of PrP-Sc in nictitating membrane lymphoid tissue of sheep. Vet Rec 142:489-491, 1998 72. O'Rourke KI, Holyoak GR, Clark WW, et al: PrP genotypes and experimental scrapie in orally inoculated Suffolk sheep in the United States. J Gen Virol 78:975-978, 1997 73. O'Rourke KI, Melco RP, Mickelson JR: Allelic frequencies of an ovine scrapie susceptibility gene. Anim Biotech 7:155-162, 1996 74. Palmer AC: Distribution of vacuolated neurons in brains of sheep affected with scrapie. J Neuropathol Exp Neurol 19:102-110, 1960 75. Parry HB: Scrapie: A transmissible and hereditary disease of sheep. Heredity 17:75105, 1962 76. Parry HB: Elimination of natural scrapie in sheep by sire genotype selection. Nature 277:127-129, 1979 77. Pattison IH: Fifty years with scrapie: A personal reminiscence. Vet Rec 123:661-666, 1988 78. Pattison IH, Hoare MN, Jebbett IN, et al: Spread of scrapie to sheep and goats by oral dosing with foetal membranes from scrapie-affected sheep. Vet Rec 90:465-468, 1972 79. Prusiner SB: Novel proteinaceous infectious particles cause scrapie. Science 216:136144, 1982 80. Race R, Ernst D, Jenny A, et al: Diagnostic implications of detection of proteinase K-resistant protein in spleen, lymph nodes, and brain of sheep. Am J Vet Res 53:883-889, 1992 81. Race R, Sutton D: Scrapie infectivity and proteinase K-resistant prion protein in sheep placenta, brain, spleen, and lymph node: Implications for transmission and antemortem diagnosis. J Infect Dis 178:949-953, 1998 82. Rubenstein R, Merz PA, Kascsak RJ, et al: Detection of scrapie-associated fibrils (SAF) and SAF proteins from scrapie-affected sheep. J Infect Dis 156:36-42, 1987 83. Safar }WH, Itri V, Groth D, et al: Eight prion strains have PrP(Sc) molecules with different conformations. Nat Med 4:1157-1165, 1998 84. Schaller 0, Fatzer R, Stack M, et al: Validation of a Western immunoblotting procedure for bovine PrpSc detection and its use as a rapid surveillance method for the diagnosis of bovine spongiform encephalopathy (BSE). Acta Neuropathol (BerI) 98:437-443, 1999 85. Schmerr MJ, Jenny A: A diagnostic test for scrapie-infected sheep using a capillary electrophoresis immunoassay with fluorescent-labeled peptides. Electrophoresis 19:409-414, 1998 86. Schreuder BEC, van Keulen LJM, Vromans MEW, et al: Tonsillar biopsy and PrPSc detection in the preclinical diagnosis of scrapie. Vet Rec 142:564-568, 1998 87. Silverman GL, Qin K, Moore RC, et al: Doppel is an N-glycosylated, glycosylphosphatidylinositol-anchored protein. J BioI Chern 275:26834-26841,2000 88. Taylor DM: Inactivation of transmissible degenerative encephalopathy agents: a review. Vet J 159:3-4, 2000 89. Telling GC, Scott M, Mastrianni J, et al: Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein. Cell 83:79-90, 1995 90. Thorgeirsdottir S, Sigurdarson S, Thorisson HM, et al: PrP gene polymorphism and natural scrapie in Icelandic sheep. J Gen Virol 80:2527-2534, 1999 91. Tranulis MA, Osland A, Bratberg B, et al: Prion protein gene polymorphisms in sheep with natural scrapie and healthy controls in Norway. J Gen Virol 80:1073-1077, 1999 92. van Keulen LJM, Schreuder BEC, Meloen RH, et al: Immunohistochemical detection of prion protein in lymphoid tissues of sheep with natural scrapie. J Clin Microbiol 34:1228-1231, 1996 93. van Keulen LJM, Schreuder BEC, Meloen RH, et al: Immunohistochemical detection and localization of prion protein in brain tissue of sheep with natural scrapie. Vet Pathol 32:299-308, 1995 94. van Keulen LJM, Schreuder BEC, Vromans MEW, et al: Scrapie-associated prion protein in the gastro-intestinal tract of sheep with natural scrapie. J Comp Pathol 121:55-63, 1999 95. Wells GAH, Scott AC, Johnson CT, et al: A novel progressive spongiform encephalopathy in cattle. Vet Rec 121:419-420, 1987
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96. Westaway D, Zuliani V, Cooper CM, et al: Homozygosity for prion protein alleles encoding glutamine-In renders sheep susceptible to natural scrapie. Genes Devel 8:959-969, 1994 97. Will RG, Ironside JW, Zeidler M, et al: A new variant of Creutzfeldt-Jakob disease in the UK. Lancet 347:921-925, 1996 98. Williams ES, Young S: Spongiform encephalopathies in cervidae. Rev Sci Tech Office Intemationale des Epizootics 11:551-567, 1982 99. Wineland NE, Detwiler L, Salmon D: Epidemiologic analysis of reported scrapie in sheep in the United States: 1,117 cases (1947-1992). J Am Vet Med Assoc 212:713718, 1998 100. Wyatt JM, Pearson GR, Smerdon T, et al: Spongiform encephalopathy in a cat. Vet Rec 126:513, 1990 101. Zlotnik I: The pathology of scrapie: A comparative study of lesions in the brain of sheep and goats. Acta Neuropathol (Bed) suppl 1:61-70, 1962
Address reprint requests to Katherine I. O'Rourke, PhD USDA, ARS, ADRU 3003 ADBF, WSU Pullman, WA 99164-6630 e-mail: [email protected]
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OVINE SCRAPIE New Tools for Control of an Old Disease Katherine 1. O'Rourke, PhD
Ovine scrapie was described as early as 1732, and fascinating accounts of research on this enigmatic disorder have been published in nearly every decade of the 20th century. The transmissible nature of scrapie was demonstrated in 1936, and the resistance of the agent to inactivation by phenol, chloroform, formalin, heat, or irradiation was established within the next 15 years. The occurrence of scrapie in particular bloodlines was convincing enough to warrant consideration of scrapie as a genetic disease for a short time. Subsequent studies demonstrated that scrapie is not an inherited disease; rather, susceptibility to scrapie is under genetic control and typically only sheep of particular genetic backgrounds develop disease in infected flocks. The hallmark microscopic lesion, vacuolated neurons in the medulla oblongata, was described in the late 1800s. Although pathologists continue to debate the diagnostic value of a lesion that occurs at low levels in uninfected sheep, the designation transmissible spongiform encephalopathy (TSE) was applied to scrapie and subsequently to similar neurodegenerative disorders of other mammalian species. Scrapie research intensified in the early 1950s after the importation of infected sheep by Canada, the United States, and Australia. The role of a nucleic acid free agent (the "proteinonly hypothesis") was suggested by several lines of study, and the extensive studies of Stanley Prusiner and his group on an infectious
This work was supported by the Agricultural Research Service, US Department of Agriculture, Grant CWU 5348-32000-015-00D. All material in this article is in the public domain with the exception of any borrowed tables.
From the US Department of Agriculture, Animal Disease Research Unit, Pullman, Washington
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protein (the prion) culminated in the 1997 Nobel Prize. This was the second such prize to be awarded for work with the TSEs. Carleton Gajdusek's work with kuru, a TSE occurring among a Stone Age tribe in Papua New Guinea, demonstrated that a human TSE could be transmitted experimentally to nonhuman primates. These transmission experiments were suggested by a letter to Dr. Gajdusek from the eminent veterinary neuropathologist William Hadlow, who remarked on the similarities between kuru and ovine scrapie. Kuru was originally thought to be transmitted through cannibalism; subsequent review of the primary data is less exotic and suggests that transmission could occur through inadvertent oral or parenteral exposure to neural tissues during funeral rituals. Oral exposure may be a major route of transmission in natural ovine scrapie after contamination of pastures, barns, feed, or water with a transmissible agent shed in the placenta or fetal fluids of an infected dam. Chronic wasting disease, a TSE of deer and elk, was reported in 1980 and now is endemic in free-ranging cervids in a small area in the western United States and on game farms in several states. Oral exposure to cattle feed contaminated with a heat-resistant transmissible agent probably contributed to epidemic levels of a new bovine spongiform encephalopathy in the United Kingdom, first reported in the late 1980s. Tragically, bovine spongiform encephalopathy is almost certainly the cause of a novel TSE of humans, variant Creutzfeldt-Jakob disease, a disorder currently diagnosed primarily in young adults. The emergence of variant Creutzfeldt-Jakob disease has lent urgency to research on the TSEs and efforts to eradicate scrapie, one of its oldest members. The literature on scrapie and the other TSEs is vast, contentious, and often contradictory. The references in this article are representative of the entire body of work, and the reader is referred to scientific databases for listings of primary research publications. From the clinician's point of view, the most important questions concern transmission, diagnosis, and control of scrapie. Although definitive answers to these questions are not yet known, regulatory agencies and producers are faced with the task of eradicating scrapie while maintaining an economically viable sheep industry in the United States and abroad. This article contains brief descriptions of the TSEs and the proposed causative agent, the associated pathogenesis and pathology, and possible routes of transmission to give clinicians the background for understanding this rapidly changing field of scientific inquiry, the emerging disorders in the TSE family, and new techniques being applied to the diagnosis and control of scrapie. THE FAMILY OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES
Scrapie is the prototype for a diverse group of fatal neurodegenerative disorders classified as TSEs. TSEs of ruminant animals include ovine scrapie, first reported in 173277 and now endemic in many parts of the
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world; bovine spongiform encephalopathy of domestic cattle and captive nondomestic ruminants,95 apparently a food-borne disorder related to the practice of incorporating rendered livestock byproducts into cattle feed; and chronic wasting disease of deer and elk in the United States,9S of unknown etiology but unrelated to bovine spongiform encephalopathy-contaminated feed. TSEs of carnivores include transmissible mink encephalopathy of farm-raised mink,39 of uncertain etiology, and feline spongiform encephalopathy of domestic and nondomestic cats,100 probably originating from exposure to bovine spongiform encephalopathy. The human TSEs include a number of familial disorders associated with point mutations in the PRPN gene. 16 Creutzfeldt-Jakob disease refers to several of these familial diseases, as well as to sporadic TSEs unrelated to PRPN polymorphisms, and to iatrogenic disease associated with corneal transplants, dura mater grafts, or parenteral inoculation with pituitary extracts from inapparently infected donors. Kuru, a human TSE of the Fore tribe of Papua New Guinea, was apparently transmitted by exposure to an infectious agent during handling of tissues during funeral rituals. Variant Creutzfeldt-Jakob disease97 is a newly emerging human TSE associated with exposure to a transmissible agent related to bovine spongiform encephalopathy.13 Variant Creutzfeldt-Jakob disease differs from sporadic, iatrogenic, and familial CreutzfeldtJakob disease in the age distribution of affected individuals, clinical course, diagnostic signs, pathogenic lesion pattern, and distribution of the disease-associated prion protein in extraneural tissues. 56 OVINE SCRAPIE, THE PROTOTYPE TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHY
Most of the reported cases of scrapie in US sheep occur in 3- to 5year-old ewes, although the disease occurs in older ewes, in rams, and in wethers.99 Clinical scrapie occurs at an earlier age (approximately 24 months) in sheep with differing genetic backgrounds in other endemic regions. s,26 Early signs may include behavioral changes (apprehension, failure to stay with the flock, reluctance to approach feed bins) followed by weight loss, incoordination, and loss of wool through rubbing or biting. In some sheep, central nervous system signs become more pronounced over the course of several weeks and include high stepping of the front legs, hopping of the back legs when moving quickly, or inability to rise. Some sheep with prion accumulation in the brain are found dead after stressful events such as shearing, vaccinations, or transport. Transmission
The natural routes of transmission are not known. The incidence of scrapie in lambs born to infected ewes is high and the risk remains high for 60 to 90 days after lambing. 21,44 Unrelated lambs and, to a lesser
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extent, adult sheep and goats may also become infected if housed with infected ewes during lambing. A transmissible agent and the prion protein have been detected in placenta and fetal membranes. 68, 78, 81 Infection may occur through the oral route when infectious tissues or fluids contaminate the bedding, feed, water, or pen surfaces. The agent is probably taken up by lymphoid tissues in the gut/a, 94 disseminated to other lymphoid tissues,35 and eventually spread to the brain through the nervous supply from the nodes. Alternatively, the pathogenic agent may be taken up directly by the enteric nervous system and transported by axonal flow through the vagus nerve to its dorsal motor nucleus,3,40 the site of early prion accumulation in the central nervous system. Causative Agent: The Prion, an Infectious Protein?
The transmissible agent in infectious tissue extracts shows remarkable resistance to inactivation by heat, nucleases, irradiation, and chemical disinfectants (reviewed elsewhere).77,88 The proteinaceous nature of the transmissible agent was suggested by these early inactivation trials. Ownership of the concept of an infectious protein (later termed the prion) is divided among several mathematicians, biochemists, and virologists, but the extensive studies initiated by Stanley Prusiner at the University of California at San Francisco provide the theoretical framework for thousands of additional studies conducted in laboratories around the world. The prion theory79 remains controversial, but no unequivocal demonstration of a conventional microorganism, virus, or subviral particle has yet been made. Prions (PrPs) are expressed as normal cellular sialoglycoproteins, ubiquitously distributed in mammalian tissues. 6, 67 PrP is encoded by the highly conserved PRNP gene,! a single-copy gene with a newly described ancestral copy (PrP doppel).87 The normal cellular isoform is termed PrP-C or PrP-sen to identify it as a normal cellular protein sensitive to digestion with proteases. The abnormal isoform is PrP-Sc (as a generic term for the protein in the disease-associated conformation), or is referred to by disease-specific designations (PrP-Sc, PrP-CJD, PrP-BSE, PrP-CWD; CJD = Creutzfeldt-Jakob disease; BSE = bovine spongiform encephalopathy; CWD = chronic wasting disease) or the biochemical definition PrP-res, to show its relative resistance to proteinases. PrP-Sc is the major component of infectious extracts and may represent the transmissible agent,79 either alone or with yet-undefined cofactors.89 Molecular modeling and in vitro systems7, 17,43,58 suggest that PrPSc molecules, generated endogenously in familial cases or introduced exogenously in transmitted cases, form aggregates with PrP-C. A biochemical trigger or seeding event initiates a structural change in some regions of the PrP-C molecules from the native alpha-helical configuration to the beta sheet folds characteristic of PrP-Sc. This conversion in secondary structure results in characteristic changes in detergent solubility, partial resistance to digestion by some proteinases, and partial resis-
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tance to denaturation by formalin, irradiation, or heat. The remarkably stable PrP-Sc molecules propagated in the cell can then serve as templates for further conversion of PrP-C in other cells or tissues and as the transmissible agent when shed by the infected host. Elegant experiments with gene-ablated PrP-I- "knock-out" mice reconstituted with normal PrP+ I + central nervous system tissue demonstrated that PrP-C is required for PrP-Sc accumulation and disease progression. 10, 14 Furthermore, PrP-C expression in an immune system with functional follicular dendritic cells is required for propagation of extraneural prions and neuroinvasion. s, 9,12 Amplification of the agent in lymphoid tissues is a hallmark of ovine scrapie, and accumulation of prions during the long incubation period is the basis for preclinical diagnosis of scrapie (described in a following section). Pathogenesis
The classic hallmark of scrapie is neuronal loss, although the mechanism of cell death is poorly understood. Accessory cells, including astrocytes and microglia, appear to participate in neuronal 10ss11,30 through release of soluble mediators or excitatory compounds. The direct role of PrP-Sc in pathogenesis remains controversial. Neuronal apoptosis can be induced in neural cultures by exposure to prion protein fragments. 2S, 31, 42 Conversely, accumulation of PrP-Sc in the absence of clinical disease or pathologic changes41 has been reported. The entire spectrum of prion-related molecules and pathogenic mechanisms may take decades to unravel. In the meantime, control of ovine scrapie relies heavily on detection of the PrP-Sc molecule. A better understanding of the molecular pathogenesis of scrapie in natural disease and in experimental models will be critical in the design of drugs needed for intervention in variant Creutzfeldt-Jakob disease and familial TSEs. Diagnosis
Scrapie is a degenerative disease of the central nervous system with no evidence of inflammation or an immune response. Diagnosis of ovine scrapie has traditionally been made after the appearance of suspect clinical signs, with confirmation by light microscopic examination of the brainstem. Histologic diagnosis, however, is based on detection of three nonspecific neuropathologic changes, which may not be apparent during early clinical disease or in autolyzed tissue and may be equivocal even at the clinical phase in some breeds. After development of the prion hypothesis, diagnosis has been supplemented by biochemical detection of PrP-Sc using immunohistochemistry and Western immunoblot analysis. Although examination of the brain remains the gold standard for diagnosing scrapie in sheep with clinical signs, detection of PrP-Sc may be a more accurate approach for diagnosis of preclinically infected
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sheep. The sensitivity of diagnostic testing could be further increased by inclusion of lymphoid tissues of the head and the gut, where PrP-Sc accumulation precedes central nervous system prion propagation by more than a year. Diagnosis by Histology
Scrapie has classically been diagnosed by postmortem examination of the brain for a combination of spongiform change, neuronal degeneration with or without vacuolation, and astrocytosis. 28, 29, 34, 35,74 The extent of spongiform change in the gray matter neuropil varies among breeds of sheep and may be absent or barely detectable. 34 Neurons with large vacuoles in the perikaryon are observed at low levels in normal sheep, and inclusion of this lesion in the diagnostic criteria is based on distribution and extent. 28,101 Astrocytosis, including both hypertrophy and hyperplasia of astrocytes, is a nonspecific reactive response. Although its diagnostic value is debated,28, 29 astrocytosis can be helpful in establishing a histologic diagnosis when the other two signs are minima1. 34 Immunohistochemistry of the Brain
Immunohistochemistry is a useful adjunct to histology because the assay is conducted on sections from the same formalin-fixed, paraffinembedded specimens used for conventional pathology. The earliest accumulation of PrP-Sc in the brain occurs in the dorsal motor nucleus of the vagus nerve, a small structure in the medulla oblongata at the level of the obex. Accurate diagnosis of early cases depends of careful collection of the medulla and proper orientation of the tissue in the paraffin block. There are currently no antibodies that distinguish PrP-Sc from its normal cellular precursor PrP-C in immunohistochemistry; however, formalin fixation, formic acid pretreatment, and mild protease digestion eliminate PrP-C immunoreactivity in many detection systems. 15, 27, 64, 70, 93 Antigen retrieval using high temperatures and either neutral or acid pH buffer increases the sensitivity of PrP-Sc immunohistochemistry on ovine tissues. 37, 38, 65 Immunohistochemistry assays for PrP-Sc using polyclonal rabbit antisera and mouse monoclonal antibodies 69-7l have been described. Polyclonal antisera have the potential advantage of reacting with multiple epitopes on the prion protein but may be lacking in titer, specificity, and quantity. Panels of monoclonal antibodies reacting to distinct sites on the prion protein provide a reasonable compromise between the need for broadly specific reagents for testing sheep of diverse genetic backgrounds and the need for defined specificity and unlimited quantities for large-scale standardization and validation of diagnostic tests. PrP-C co-localizes with PrP-Sc in tissues from infected sheep; the relative amounts of PrP-C and PrP-Sc may vary with breed, age, length of infection, and genotype. The efficacy of pretreatment steps to eliminate the reactivity of PrP-C and the antigen retrieval steps to enhance
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PrP-Sc detection must be validated in sheep of various breeds and genotypes for each combination of primary antibody and detection system. Comparison studies under way in the United States and internationally will result in standardized protocols for detection of PrP-Sc in ruminant tissues. Other Diagnostic Tests for Ruminant Transmissible Spongiform Encephalopathies
If unfixed tissue is available, supplementary diagnostic techniques include Western immunoblot detection of proteinase K-resistant prion protein2, 18,24,62,66,80,82 or detection of scrapie-associated fibrils by electron microscopy.63 Western blot analysis is based on the relative protease resistance of the PrP-Sc protein core. Tissue homogenates from uninfected sheep typically show three bands reactive with anti-PrP antibodies. These bands represent the unglycosylated protein precursor and the successively larger mono- and diglycosylated PrP molecules. Proteinase K digestion hydrolyzes PrP-C into small fragments undetectable under these assay conditions. Proteinase K treatment of PrP-Sc cleaves only an amino terminal fragment, slightly reducing the apparent molecular weight of each of the three bands. Comparison of banding patterns before and after digestion is used to establish a diagnosis. Careful control of the proteinase K digestion step is required to verify that proteinase K digestion is complete despite differences in the amount of tissue in the homogenate, the activity of the proteinase K enzyme lot, or endogenous serine proteinase inhibitors commonly found in some ovine tissues. This type of Western blot test in a kit format (Check Test, Prionics, Zurich, Switzerland)84 is validated for diagnosis of bovine spongiform encephalopathy using brain tissue collected from slaughter cattle in Switzerland and France. In tissues with sparsely distributed PrP-Sc, an enrichment step exploiting the relative insolubility of PrP-Sc in detergent solutions is typically incorporated. 8o,82 Novel test techniques with increased sensitivity or higher throughput are in the development stages. 60, 83, 85 Postmortem Diagnosis of Scrapie' Using Lymphoid Tissues
Accumulation of the transmissible agent in lymphoid tissues precedes deposition in the central nervous system,35 so assay of lymphoid tissues for PrP-Sc is the basis for early diagnostic testing of sheep.55, 80, 92 PrP-Sc was detected by immunohistochemistry assay in a consistently high percentage of lymphoid follicles in 98% (54 of 55) of palatine tonsil samples collected at necropsy from scrapie-infected sheep in the Netherlands.92 Similar observations were made in a smaller study in which tonsil was available from 47 US sheep (31 uninfected and 16 infected animals).69 PrP-Sc was detectable in tonsil tissue of infected sheep at 4 to 8 months of age in flocks with a typical incubation time of 24 months, and at approximately 9 to 14 months in scrapie-infected US sheep with a typical incubation time of 32 to 48 months. The medial
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retropharyngeallymph node is nearly as informative as tonsil, although additional sections must be examined in early cases because PrP-Sc is not distributed uniformly throughout this relatively large lymph node. Additional candidate tissues include the gut-associated lymphoid tissues (ileal Peyer's patches and ileocecal lymph node), presumably the sites of the earliest uptake and propagation of PrP-Sc in orally exposed sheep. In immunohistochemistry studies in the Netherlands and the United States and in an earlier rodent bioassay study,36 PrP-Sc or a transmissible agent was detected in brain but not in tonsil. The value of lymphoidbased tests for early diagnosis will depend in part on an emerging understanding of the pathogenesis of ovine scrapie, including the effects of breed, age, genotype, dose and route of infection, and scrapie strain on distribution of PrP-Sc in peripheral lymphoid tissues. Based on the findings cited, a reliable tissue set for preclinical postmortem diagnosis includes the medulla oblongata at the level of the obex (to include the dorsal motor nucleus of the vagus), tonsil or retropharyngeal lymph node, and ileal Peyer's patches or ileocecal lymph node. Validation of standardized methods is under way in several countries. The US effort includes long-term quarantine of high-risk sheep, with antemortem and postmortem testing by immunohistochemistry and postmortem histology, Western immunoblot, and bioassay of various neural and extraneural tissues in susceptible rodents and ruminants. Antemortem Preclinical Diagnosis by Immunohistochemistry of Peripheral Lymphoid Tissues
Antemortem diagnosis of scrapie was described by Schreuder et al86 using 4-mm samples of tonsillar tissue collected with biopsy forceps using full or partial anesthesia in larger sheep and manual restraint in younger animals. Dr. Steven Parish, a clinician at Washington State University, examined alternative sites of germinal center formation that could be sampled under local anesthesia with disposable instrumentation. Lymphoid tissue on the bulbar surface of the nictitating membrane (the "third eyelid" lymphoid tissue) can be sampled using inexpensive single-use surgical instruments after application of a topical ophthalmic anesthetic.71 This test has been evaluated in clinically affected sheep, in sheep with no exposure to scrapie, and in clinically normal sheep with preclinical disease (Table 1).71 The third-eyelid test is useful in sheep after 14 months of age, assuming that infection is acquired during the perinatal period. Sheep with positive eyelid biopsy samples developed scrapie between 3 and 20 months after testing, when they reached 3 to 4 years of age. The third-eyelid test has completed the first two stages of diagnostic test validation, as described in the Office International des Epizooties Manual of Standards for Diagnostic Tests,57 and has an estimated sensitivity of 88% and a specificity of greater than 97% (Table 1). The test is currently the focus of large-scale validation studies in the United States and Canada.
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Table 1. IMMUNOHISTOCHEMISTRY ASSAY OF THIRD EYELID LYMPHOID TISSUE FOR DIAGNOSIS OF OVINE SCRAPIE Scrapie Status
Number
Eyelid Positive*
Eyelid Negative*
Confirmed scrapie, t clinically suspectt Uninfected sheep§: Sheep with suspect eNS signs (n = 7), clinically normal sheep exposed to scrapie (n = 120), or sheep with no exposure to scrapie (n = 48) Confirmed scrapie: Clinically normal when tested at necropsy (n = 13) or quarantined until progression to clinical disease 3 to 20 months after biopsy (n = 28)
42 175
41 1
1 174
41
36
5
*Results of immunohistochemistry assay performed with MAb F89/160.1.5. tScrapie-infected sheep showed PrP-Sc immunostaining in medulla oblongata at the level of the obex (US samples) or routine histopathology of brain (UK samples). tClinical signs included wool rubbing, weight loss, and ataxia. §Scrapie-uninfected sheep showed no lesions characteristic of scrapie (UK samples) or no PrP-Sc detectable in brain, tonsil, retropharyngeal lymph node, or submandibular lymph node (US samples, n = 127) or had no reported exposure to scrapie in the flock for 5 years (n = 48). Data from O'Rourke KI, Baszler TV, Besser TE, et al: Preclinical diagnosis of scrapie by immunohistochemistry of the third eyelid lymphoid tissue. J Clin Microbio138:3254-3259, 2000.
GENETIC SUSCEPTIBILITY
Scrapie is a transmissible disease in which incubation time and susceptibility to clinical disease are under genetic control. The association between scrapie and particular PRNP alleles has been described for many breeds of sheep under a wide variety of field and experimental conditions. Genetic testing of breeding stock and selection for animals carrying the genes associated with low susceptibility may reduce the risk of scrapie.
The Ovine Prion Gene
The ovine PRNP gene contains the genetic blueprint for the 254 amino acids of the prion protein. One codon (three DNA bases) defines each amino acid. The DNA sequence is variable (polymorphic) at several sites, and each alternative sequence (allele) is named by the corresponding amino acids at these sites, using the single-letter biochemical abbreviation for each amino acid. The codons most closely associated with susceptibility and incubation time are those encoding amino acids 136 (A or V), 154 (R or H), and 171 (Q, R, or H). Predominant alleles for various breeds of sheep in the United Kingdom and the United States are summarized in Table 2.19, 61, 73 Each sheep inherits two copies of the gene, one from each parent. The alleles may be identical (in a homozygous animal) or may differ (in a heterozygous animal), and the combina-
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Table 2. DISTRIBUTION OF PRNP ALLELES REPORTED IN SHEEP IN THE UNITED STATES AND THE UNITED KINGDOM Breeds
Predominant Alleles
ARQ and ARR ARQ, ARR, and VRQ ARQ, AHQ, VRQ, ARR, and ARH ARQ, ARR, and AHQ ARQ, ARR, AHQ, and VRQ
UK Suffolk, UK Cotswold, US Dorset UK Poll Dorset, UK Border Leicester US Suffolk,* UK Texel UK Bluefaced Leicester UK Cheviot, US Cheviot, UK Swaledale, UK Welsh Mountain
*Surveys of US Suffolk sheep71 (unpublished data) show allelic frequencies of approximately 70% ARQ, 25% ARR, and 5% VRQ, AHQ, and ARH. The most common diploid genotypes are ARQ/ ARQ (approximately 45% of surveyed sheep in flock with no selection for scrapie susceptibility), ARQ/ ARR (approximately 35%), ARR/ ARR (approximately 10%), VRQ/ ARQ or VRQ/ ARR (approximately 5%); all other genotypes (approximately 5%).
tion of both alleles is referred to as the animal's scrapie susceptibility
genotype. Association of Scrapie with Ovine Genetics
Forty years ago, Parry75,76 used classical breeding studies in unsuccessful attempts to demonstrate that scrapie is a familial disease. Subsequent studies demonstrated the inheritance of disease susceptibility rather than the disease itself.20, 22, 45, 46, 48 The association between specific alleles of the PRNP gene and clinical scrapie has been demonstrated under a wide variety of field and experimental conditions in several breeds.* Variation at codon 154 modulates incubation time in some breeds and is associated with some reduction in susceptibility in other breeds. 19,90 Susceptibility is more clearly associated with variation at codons 136 and 171 in most breeds examined. In breeds in which the VRQ allele occurs at high frequency, scrapie is found in sheep with one or two VRQ alleles. Sheep lacking this allele resist experimental challenge and are at low risk of scrapie. In breeds generally lacking the VRQ allele, scrapie susceptibility is controlled by polymorphisms at codon 171. Sheep homozygous for 171Q are susceptible to experimental and natural scrapie; sheep with at least one ARR allele rarely develop clinical disease or PrP-Sc accumulation in brain or lymphoid tissues. Potential Drawbacks to Genetic Selection
Based on the numerous studies cited previously, relative genetic susceptibility to scrapie in sheep homozygous or heterozygous for the *References 4, 8, 22, 23, 32, 33, 47, 49-54, 59, 61, 68, 72, 90, 91, and 96.
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VRQ allele or homozygous for the ARQ allele is well accepted. The relative scrapie "resistance" of sheep with the genotypes ARQ/ ARR or ARR/ ARR is less well defined. Scrapie has been reported in a small number of sheep with these genotypes. 54,91 There is also some concern that heterozygous ARQ/ ARR sheep may develop a silent carrier state, shedding the transmissible agent while remaining clinically normal. Data from a large field study in France23 failed to demonstrate such a carrier state in heterozygous ARQ/ ARR sheep, and PrP-Sc has not yet been detected in brain or lymphoid tissues of ARR sheep in studies in this laboratory (unpublished data, 1990-2000). Studies monitoring sheep for a possible carrier state are in progress in the United States, the United Kingdom, and Europe. Scrapie Susceptibility Genetics and Control Programs
The United Kingdom has proposed a scrapie risk reduction program based on increasing the frequency of the ARR allele in purebred stock. Rams are scored for relative susceptibility by diploid genotype, ranging from highly susceptible (RS) through relatively resistant (R1) (Table 3). The relative risk of scrapie in first-generation progeny is weighted more heavily than relative risk to the breeding ram in this scheme. Only rams with the lower susceptibility scores R1 and R2 are to be used in the proposed UK National Scrapie Plan. In the United States, one state (Michigan) has proposed a scrapie control program based on gradual transition to R1 genotypes in breeding stock, and a second state (Washington) limits the movement of rams with the RS genotype. The contribution of scrapie susceptibility genotyping in scrapie eradication is under investigation in a large-scale field trial in the United States. PREVENTION AND CONTROL
The risk of scrapie can be reduced through a coordinated program of flock management, genetics, and preclinical testing. Purchase of healthy animals is the cornerstone of a good management program. In the United States, state and federal programs such as the US Department of Agriculture Voluntary Scrapie Flock Certification Program can provide flock owners with a source of sheep from flocks monitored for scrapie. Purchased ewes from other flocks, particularly ewes of the high-susceptibility genotype, should be considered a potential risk. Good lambing records, including date and month of lambing with information on housing of ewes during this period, should be maintained and kept for 6 years. Provision of separate lambing quarters for genetically susceptible purchased ewes, with eyelid testing at least 14 months after arrival and culling of test-positive ewes, may reduce the risk of transmission to other animals in the flock. Good sanitation, including prompt disposal of placental tissue, fetal membranes, and contaminated bedding, with
POSED SCORING SYSTEM FOR SELECTING BREEDING RAMS BASED ON ESTIMATED RISK OF SCRAPIE IN THE RA -GENERATION PROGENY Risk of Scrapie Infection in Predominant Genotypes
ARR/ARR ARRlAHQ or AHQ/ AHQ ARRlARQ ARRIARH
ARQ/AHQ ARH/ARH ARQ/ARH ARQ/ARQ ARR/VRQ AHQ/VRQ VRQ combined with any other allele
Ram Type in Propos UK National Scrapie P
Ram
First Generation
Very low Low Low
Low Low Variable (dependent on genotype of ewe)
Type I Type II genotypes in bold Type II genotypes in bold
Medium
Generally higher than progeny of R3 rams
Only used during the first 2 yea Type I and Type II rams are not
High
Generally higher than progeny of R3 and R4 rams
Not allowed
sk of scrapie in ram and first-generation progeny from very low (R1) to very high (R5) after natural exposure to scrapie in the flock. I and Type II rams could be used for breeding stock; progeny of Type II rams would require continued genetic testing. m Dawson M, Hoinville LJ, Hosie BD, et al: Guidance on the use of PrP genotyping as an aid to the control of clinical scrapie. Vet Rec 142:623-
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cleaning of implements and washable surfaces with a solution of 40% household bleach88 should reduce the level of the infectious agent in lambing quarters. Eyelid testing of live sheep may be recommended or required by regulatory agencies after possible exposure to scrapie. Postmortem testing of brain, tonsil, and ileocecal lymph node of sheep dying of unknown causes is recommended, particularly for high-susceptibility sheep purchased from flocks of unknown scrapie status. A longterm, cost-effective preventive approach is identification of candidate rams with good breeding potential, followed by codon 171 testing and selection of 171RR (Type I) or 171QR (Type II) rams from that group. Fully susceptible 171QQ rams with superior breeding characteristics can be used with 171RR ewes to increase the supply of rams for this program. Large-scale surveillance of slaughter samples and random live animal testing at fairs and sales may eventually be used to identify infected flocks. An integrated program of management, preclinical testing, and genetics can prevent the introduction and spread of scrapie within flocks and may eventually contribute to the eradication of scrapie in the United States.
VETERINARIANS' ROLES IN SCRAPIE ERADICATION
Scrapie eradication in the United States involves veterinarians as research scientists, pathologists, diagnosticians, and regulatory personneL Most importantly, veterinary clinicians represent the interface between producers and a bewildering array of rapidly changing recommendations, regulations, and diagnostic tests. In the early stages of an eradication program, an increasing number of infected flocks will be identified by these tests, with subsequent losses of sheep that are often known to the producer by name as well as eartag number or sale price. The veterinarian will be charged with educating producers about the need for stringent control programs, but he or she will also serve by relaying to the regulatory bodies the need for compassion in the development and conduct of a scrapie eradication program. Resources for veterinarians can be found through continuing education programs and through the US Department of Agriculture / Agricultural Research Service (Animal Disease Research Unit: http://pwa.ars. usda.gov / adru) or Animal and Plant Health Inspection Service (Veterinary Services Scrapie Control Programs: http:/ / www.aphis.usda. gov /vs/scrapie). Information on the British-proposed National Scrapie Plan based on ram genetics is found at http://www.maff.gov.uk/ animalh/bse /bsescience / scrapie / nsp / nsp.htmL Commercial genotyping laboratories in the United States are Livestock Molecular Research and Development (Monticello, IL; telephone: 217-762-3094) and GeneCheck (Fort Collins, CO; telephone: 800-822-6740).
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Address reprint requests to Katherine I. O'Rourke, PhD USDA, ARS, ADRU 3003 ADBF, WSU Pullman, WA 99164-6630 e-mail: [email protected]