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Western immunoblot analysis of U.S. Nor98 scrapie-positive sheep. PrP Sc in proteinase K-digested brain tissue homogenates. A, lane 1: U.S. Nor98 case 1; lane 2: molecular marker; 3 5 classic scrapie negative control; lane 4: Nor98 positive control. B, lane 1: classic scrapie positive control; lane 2: molecular marker; lanes 3, 4: U.S. Nor98 case 2. C, lane 1: classic scrapie positive control without PNGase; lane 2: classic scrapie positive control with PNGase treatment; lane 3: U.S. Nor98 case 2 without PNGase; lane 4: U.S. Nor98 case 2 with PNGase treatment. D, lane 1: classic scrapie positive control; lane 2; bovine spongiform encephalopathy (BSE) positive control; lane 3: molecular marker; lane 4: Nor98 positive control; lane 5: molecular marker; lane 6: U.S. Nor98 case 4; lane 7: molecular marker. E, lane 1: molecular marker; lane 2: classic scrapie positive control; lane 3: BSE positive control; lane 4: U.S. Nor98 case 5; lane 5: molecular marker. Arrow in each panel represents 30 kDa.
Source publication
A distinct strain of scrapie identified in sheep of Norway in 1998 has since been identified in numerous countries throughout Europe. The disease is known as Nor98 or Nor98-like scrapie, among other names. Distinctions between classic scrapie and Nor98 scrapie are made based on histopathology and immunodiagnostic results. There are also differences...
Contexts in source publication
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... white matter (Fig. 1D). PrP Sc immunolabeling was absent from the dorsal motor nucleus of the vagus nerve and lymphoid tissue. ELISA results were positive when using cerebellum by both the TSE sheep and goat kit k and the scrapie antigen kit l (Table 1). Western blot results included multiple protein bands with an unglycosylated band at ,15 kDa (Fig. 2B, lanes 3 and 4). Digestion with PNGase after proteinase K treatment resulted in 3 unglycosy- lated bands (Fig. 2C, lane 4) comigrating with the lower 3 bands of the untreated sample (Fig. 2C, lane 3). A classic scrapie sample treated under identical conditions yielded a single band comigrating with the lowest band in the untreated sample (Fig. 2C, ...
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... tissue. ELISA results were positive when using cerebellum by both the TSE sheep and goat kit k and the scrapie antigen kit l (Table 1). Western blot results included multiple protein bands with an unglycosylated band at ,15 kDa (Fig. 2B, lanes 3 and 4). Digestion with PNGase after proteinase K treatment resulted in 3 unglycosy- lated bands (Fig. 2C, lane 4) comigrating with the lower 3 bands of the untreated sample (Fig. 2C, lane 3). A classic scrapie sample treated under identical conditions yielded a single band comigrating with the lowest band in the untreated sample (Fig. 2C, lanes 1 and 2). All sheep in this USDA-quarantined flock were tested for scrapie. No additional cases of Nor98 ...
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... TSE sheep and goat kit k and the scrapie antigen kit l (Table 1). Western blot results included multiple protein bands with an unglycosylated band at ,15 kDa (Fig. 2B, lanes 3 and 4). Digestion with PNGase after proteinase K treatment resulted in 3 unglycosy- lated bands (Fig. 2C, lane 4) comigrating with the lower 3 bands of the untreated sample (Fig. 2C, lane 3). A classic scrapie sample treated under identical conditions yielded a single band comigrating with the lowest band in the untreated sample (Fig. 2C, lanes 1 and 2). All sheep in this USDA-quarantined flock were tested for scrapie. No additional cases of Nor98 scrapie were detected, whereas 4 cases of classic scrapie were identified in ...
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... no PrP Sc immunolabeling in the dorsal motor nucleus of the vagus nerve or in lymphoid tissue. The ELISA results were positive for cerebellum by the TSE sheep and goat kit k and negative for the same tissue when using the scrapie antigen kit l (Table 1). Western blot results included multiple protein bands with an unglycosylated band at ,15 kDa (Fig. 2D, lane 6). Based on sale records, all of the animals sold at the dispersal sale went to slaughter, thus, no additional animals were available for scrapie ...
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... ELISA results were positive when using cerebel- lum by both the TSE sheep and goat kit k and the scrapie antigen kit l (Table 1). When using brainstem, ELISA was positive by the TSE sheep and goat kit k and negative by the scrapie antigen kit l (Table 1). Western blot produced multiple protein bands, including an unglycosylated band at ,15 kDa (Fig. 2E, lane 4). The flock was depopulated, and no additional cases of Nor98 or classic scrapie were identified in the adult ...
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... testing. In each case, there were multiple protein bands, including an unglycosylated band that measured ,15 kDa (Fig 2A, lane 1; B, lanes 3 and 4; D, lane 6; E, lane 4). A distinct feature in the analysis of Nor98 scrapie by Western blot testing is the presence of 3 different unglycosylated bands after PNGase treatment 15 as observed in case 2 (Fig. 2C, lane 4), whereas, with classic scrapie, just 1 band is detected (Fig. 2C, lane 2). ELISA results were positive by the TSE sheep and goat kit k for all 4 cases that included fresh tissue but were positive by the scrapie antigen kit l for only 2 of these cases (Table ...
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... band that measured ,15 kDa (Fig 2A, lane 1; B, lanes 3 and 4; D, lane 6; E, lane 4). A distinct feature in the analysis of Nor98 scrapie by Western blot testing is the presence of 3 different unglycosylated bands after PNGase treatment 15 as observed in case 2 (Fig. 2C, lane 4), whereas, with classic scrapie, just 1 band is detected (Fig. 2C, lane 2). ELISA results were positive by the TSE sheep and goat kit k for all 4 cases that included fresh tissue but were positive by the scrapie antigen kit l for only 2 of these cases (Table ...
Citations
... The term atypical scrapie has also been used to refer to prion disease in sheep and goats arising from non-native prions such as bovine spongiform encephalopathy (BSE) and chronic wasting disease (CWD). For the purposes of this manuscript, we use the term atypical scrapie to refer to only the sporadic/spontaneously occurring form of scrapie originally characterized as Nor98 scrapie [6] and known as Nor98-like scrapie for cases in the United States [7]. Classical scrapie was the first agricultural TSE to be described and has been a significant pathogen affecting sheep and goat farming in many countries for centuries. ...
... Though selective breeding has proven highly successful in efforts to eradicate classical scrapie, several cases of atypical scrapie have been detected in sheep homozygous for the ARR allele [7,25,26]. As this genotype is a foundation of resistance breeding programs, and thus will comprise an increasing proportion of domestic sheep, it is important to characterize fully the potential for atypical scrapie disease progression and transmission in these animals. ...
... In the following study, sheep bearing the ARR/ARR genotype were successfully infected with atypical scrapie isolated from a United States ARR/ARR case [7]. This allowed for a detailed examination of the disease phenotype with respect to progression and PrP Sc distribution both within the brain and in peripheral tissues. ...
Nor98-like atypical scrapie is a sporadic disease that affects the central nervous system of sheep and goats that, in contrast to classical scrapie, is not generally regarded as naturally transmissible. However, infectivity has been demonstrated via bioassay not only of brain tissue but also of certain peripheral nerves, lymphoid tissues, and muscle. This study examines placental tissue, a well characterized route of natural transmission for classical scrapie. Further, this study was conducted in sheep homozygous for the classical scrapie resistant ARR genotype and is the first to characterize the transmission of Nor98-like scrapie between homozygous-ARR sheep. Nor98-like scrapie isolated from a United States ARR/ARR sheep was transmitted to four ARR/ARR ewes via intracerebral inoculation of brain homogenate. These ewes were followed and observed to 8 years of age, remained non-clinical but exhibited progression of infection that was consistent with Nor98-like scrapie, including characteristic patterns of PrPSc accumulation in the brain and a lack of accumulation in peripheral lymphoid tissues as detected by conventional methods. Immunoblots of placental tissues from the infected ewes revealed accumulation of a distinct conformation of PrPres, particularly as the animals aged; however, the placenta showed no infectivity when analyzed via ovinized mouse bioassay. Taken together, these results support a low risk for natural transmission of Nor98-like scrapie in ARR/ARR sheep.
... 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 Australia 154 and New Zealand, 155 2 countries that are free of classical scrapie, which provides further evidence of the spontaneity of the disease. ...
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.
... 25,166 Atypical scrapie is different from classical scrapie in clinical presentation, molecular characteristics and distribution of PrP Sc within infected sheep, genotypes affected, and epidemiology. Atypical scrapie has been identified throughout Europe, 9,40,41,47,53 North America, 106,111 New Zealand, 91 and Australia. 34 The worldwide distribution with similar incidence rates where detected supports a separate etiology from classical scrapie 47 and that it is spontaneous 109 or transmits very poorly under natural conditions. ...
Scrapie is a naturally occurring transmissible spongiform encephalopathy (TSE) or prion disease of sheep and goats. Scrapie is a protein misfolding disease where the normal prion protein (PrP C) misfolds into a pathogenic form (PrP Sc) that is highly resistant to enzymatic breakdown within the cell and accumulates, eventually leading to neurodegeneration. The amino acid sequence of the prion protein and tissue distribution of PrP Sc within affected hosts have a major role in determining susceptibility to and potential environmental contamination with the scrapie agent. Many countries have genotype-based eradication programs that emphasize using rams that express arginine at codon 171 in the prion protein, which is associated with resistance to the classical scrapie agent. In classical scrapie, accumulation of PrP Sc within lymphoid and other tissues facilitates environmental contamination and spread of the disease within flocks. A major distinction can be made between classical scrapie strains that are readily spread within populations of susceptible sheep and goats and atypical (Nor-98) scrapie that has unique molecular and phenotype characteristics and is thought to occur spontaneously in older sheep or goats. This review provides an overview of classical and atypical scrapie with consideration of potential transmission of classical scrapie to other mammalian hosts.
... An atypical form of scrapie was first diagnosed in Norway in 1998 and was therefore called Nor98 (Benestad et al., 2003). Identification of the disease in Europe was made possible with the implementation of programmes of active surveillance of TSEs in small ruminants; since then, cases have been reported from almost all European countries (Belgium, Islands, France, Germany, Ireland, Norway, Portugal, Sweden, UK, Poland , Spain, Italy, the Netherlands, Finland, Denmark and Switzerland) but also in the Falkland Islands and the United States, Canada, Japan (De Bossche, 2004; Gavier-Widen et al., 2005; Epstein, 2005; Le Dur et al., 2005; Buschmann et al., 2004a; Noremark and Hope, 2006; Konold et al., 2007; Luhken et al., 2007; EFSA, 2005; Saunders et al., 2006; Loiacono et al. , 2009) Due to the low frequency of occurrence and the absence or poor representation of clinical signs, atypical scrapie is usually diagnosed during active surveillance in apparently healthy asymptomatic herds, and not in suspect clinical cases (Benestad et al., 2003;). When present in flocks, atypical scrapie affects old sheep (older than 5 years). ...
Scrapie is a neurodegenerative prion disease of sheep, goats and mouflons, belonging to the group of transmissible spongiform encephalopathies (TSEs), which affects humans as well. Even though classical scrapie has been known for over 250 years, the 1985 BSE crisis related to the advent of new forms of the Creutzfeldt-Jakob disease (vCJD) in humans imposed the implementation of rapid coercive legal measures of prevention, control and eradication of TSEs. According to the prion hypothesis, the transmissible agent is the pathological isoform (PrP Sc) of cellular prion protein (PrP C). Specific polymorphisms of the gene that encodes cell prion protein (PrnP) in sheep have been associated with resistance / natural susceptibility to the development and progression of the disease. Combinations of alleles at three adjacent codons (136 [A/V], 154 [H/R], 171 [H/Q/R]) underpin the classification of 15 possible genotypes in risk classes, applicable in selection schemes where the maximum resistance is conferred by ARR allele, and the minimum by the VRQ allele. Although, after applying these programmes, the genetic structure of sheep populations has changed favourably, genotype association studies showed that no genotype is completely resistant to the infection, including homozygote ARR / ARR. With the discovery of atypical scrapie (Nor98), it became evident that the connection between the genetics of prion protein gene polymorphisms and susceptibility to the disease must be re-evaluated individually for each breed. In scrapie monitoring and control programmes, three diagnostic categories of the disease are observed: classical scrapie, atypical scrapie and BSE scrapie in small ruminant. This review shows the chronology of progress in the fight for the eradication of TSEs in sheep, 30 years after the BSE epidemic outburst, focusing especially on the link between the molecular diagnostic forms and the genetics of the disease.
... An atypical form of scrapie was first diagnosed in Norway in 1998 and was therefore called Nor98 (Benestad et al., 2003). Identification of the disease in Europe was made possible with the implementation of programmes of active surveillance of TSEs in small ruminants; since then, cases have been reported from almost all European countries (Belgium, Islands, France, Germany, Ireland, Norway, Portugal, Sweden, UK, Poland , Spain, Italy, the Netherlands, Finland, Denmark and Switzerland) but also in the Falkland Islands and the United States, Canada, Japan (De Bossche, 2004; Gavier-Widen et al., 2005; Epstein, 2005; Le Dur et al., 2005; Buschmann et al., 2004a; Noremark and Hope, 2006; Konold et al., 2007; Luhken et al., 2007; EFSA, 2005; Saunders et al., 2006; Loiacono et al. , 2009) Due to the low frequency of occurrence and the absence or poor representation of clinical signs, atypical scrapie is usually diagnosed during active surveillance in apparently healthy asymptomatic herds, and not in suspect clinical cases (Benestad et al., 2003;). When present in flocks, atypical scrapie affects old sheep (older than 5 years). ...
Scrapie is a neurodegenerative prion disease of sheep, goats and mouflons, belonging to the group of transmissible spongiform encephalopathies (TSEs), which affects humans as well. Even though classical scrapie has been known for over 250 years, the 1985 BSE crisis related to the advent of new forms of the Creutzfeldt-Jakob disease (vCJD) in humans imposed the implementation of rapid coercive legal measures of prevention, control and eradication of TSEs. According to the prion hypothesis, the transmissible agent is the pathological isoform (PrP Sc) of cellular prion protein (PrP C). Specific polymorphisms of the gene that encodes cell prion protein (PrnP) in sheep have been associated with resistance / natural susceptibility to the development and progression of the disease. Combinations of alleles at three adjacent codons (136 [A/V], 154 [H/R], 171 [H/Q/R]) underpin the classification of 15 possible genotypes in risk classes, applicable in selection schemes where the maximum resistance is conferred by ARR allele, and the minimum by the VRQ allele. Although, after applying these programmes, the genetic structure of sheep populations has changed favourably, genotype association studies showed that no genotype is completely resistant to the infection, including homozygote ARR / ARR. With the discovery of atypical scrapie (Nor98), it became evident that the connection between the genetics of prion protein gene polymorphisms and susceptibility to the disease must be re-evaluated individually for each breed. In scrapie monitoring and control programmes, three diagnostic categories of the disease are observed: classical scrapie, atypical scrapie and BSE scrapie in small ruminant. This review shows the chronology of progress in the fight for the eradication of TSEs in sheep, 30 years after the BSE epidemic outburst, focusing especially on the link between the molecular diagnostic forms and the genetics of the disease.
... These cases were defined as atypical based on several criteria: clinical presentation, molecular characteristics of the abnormal prion protein, distribution of PrP Sc within infected sheep, genotypes of affected sheep, and epidemiology. Since 1998, additional cases have been diagnosed throughout Europe Dagleish et al. 2008;De Bosschere et al. 2007;Fediaevsky et al. 2008;Gavier-Widen et al. 2004;Loiacono et al. 2009;Mitchell et al. 2010) and one case in New Zealand (Kittelberger et al. 2010). Progressive ataxia was the predominant clinical sign in the original report (Benestad et al. 2003), but most often these cases are detected during routine diagnostic screening of older cull animals (active surveillance) where neurologic findings are absent or ill defined. ...
Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal protein-misfolding neurodegenerative diseases. TSEs have been described in several species, including bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and goats, chronicwasting disease (CWD) in cervids, transmissible mink encephalopathy (TME) in mink, and Kuru and Creutzfeldt-Jakob disease (CJD) in humans. These diseases are associated with the accumulation of a protease-resistant, disease-associated isoform of the prion protein (called PrPSc) in the central nervous system and other tissues, depending on the host pecies.
Typically, TSEs are acquired through exposure to infectious material, but inherited and spontaneous TSEs also occur. All TSEs share pathologic features and infectious mechanisms but have distinct differences in transmission and epidemiology due to host factors and strain differences encoded within the structure of the misfolded prion protein. The possibility that BSE can be transmitted to humans as the cause of variant Creutzfeldt-Jakob disease has brought attention to this family of diseases. This review is focused on the TSEs of livestock: bovine spongiform encephalopathy in cattle and scrapie in sheep and goats.
... These cases were defined as atypical based on several criteria: clinical presentation, molecular characteristics of the abnormal prion protein, distribution of PrP Sc within infected sheep, genotypes of affected sheep, and epidemiology. Since 1998, additional cases have been diagnosed throughout Europe Dagleish et al. 2008;De Bosschere et al. 2007;Fediaevsky et al. 2008;Gavier-Widen et al. 2004;Loiacono et al. 2009;Mitchell et al. 2010) and one case in New Zealand (Kittelberger et al. 2010). Progressive ataxia was the predominant clinical sign in the original report (Benestad et al. 2003), but most often these cases are detected during routine diagnostic screening of older cull animals (active surveillance) where neurologic findings are absent or ill defined. ...
Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal protein-misfolding neurodegenerative diseases. TSEs have been described in several species, including bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and goats, chronic wasting disease (CWD) in cervids, transmissible mink encephalopathy (TME) in mink, and Kuru and Creutzfeldt-Jakob disease (CJD) in humans. These diseases are associated with the accumulation of a protease-resistant, disease-associated isoform of the prion protein (called PrPSc) in the central nervous system and other tissues, depending on the host species. Typically, TSEs are acquired through exposure to infectious material, but inherited and spontaneous TSEs also occur. All TSEs share pathologic features and infectious mechanisms but have distinct differences in transmission and epidemiology due to host factors and strain differences encoded within the structure of the misfolded prion protein. The possibility that BSE can be transmitted to humans as the cause of variant Creutzfeldt-Jakob disease has brought attention to this family of diseases. This review is focused on the TSEs of livestock: bovine spongiform encephalopathy in cattle and scrapie in sheep and goats.
... The transmission of atypical scrapie has been demonstrated (28,29) but is still not fully understood. Cases of atypical scrapie have since been confirmed in the United States and Canada (30,31). According to the CFIA, 11 of 49 confirmed cases of scrapie in Canada since 2005 were identified as atypical scrapie. ...
This study analyzed sheep prion protein (PrP) genotypes of samples submitted from Ontario and other provinces of Canada to the Animal Health Laboratory at the University of Guelph, Guelph, Ontario, between 2005 and 2012. In Ontario, the proportion of scrapie-resistant sheep increased from 2005 to 2012 as evidenced by an increase in the ARR haplotype. When Canadian provinces (Alberta, Ontario, Quebec, and Nova Scotia) were compared from 2008 to 2012, a high proportion of scrapie-resistant sheep was found in all the provinces. The proportions of resistant sheep were lower in Alberta and Quebec than in Ontario and Nova Scotia. Alberta had higher proportions of susceptible sheep and a higher frequency of VRQ alleles, and Quebec had a higher frequency of the ARQ allele.
... Additional non-synonymous mutations have been reported in several breeds, (Goldmann, 2008) Norway in 1998, is a prion disease that showed distinct phenotypic characteristics compared with classical scrapie (Benestad et al., 2008). Nor98 has been identified in most European countries, in North America (Mitchell et al., 2010;Loiacono et al., 2009) and New Zealand (Kittelberger et al., 2010) with sporadic distribution. The susceptibility of sheep to this apparently spontaneous disease is also under the control of the PrP gene. ...
... IHC was performed on brainstem and retropharyngeal lymph node using a Ventana automated system, as previously described. 3 Briefly, formalin fixed, paraffin embedded tissues were sectioned at 5-µm, placed on glass slides, incubated in 95% formic acid for 5 minutes, the primary antibody (PrP-specific antibody F99/97.6.1) was applied, and detection was achieved using an alkaline phosphatase system. ...
... A BioRad ELISA test kit was used as previously described. 3 The fresh brain was friable from multiple partial freeze-thaw cycles, and tissue from grossly approximated locations was taken from the cerebrum, hippocampus, thalamus, mesencephalon, pons, trapezoid body and cerebellum. Briefly, tissue was used to make 20% homogenates, proteinase K digested, and loaded on microtiter plates coated with a PrP-specific monoclonal antibody. ...
... Western Immunoblot A BioRad kit was used for the Western immunoblot as previously described, 3 using tissue from the obex area of the brainstem. Briefly, 20% homogenates of brain tissue were proteinase K digested, PrP res was concentrated, solubilized in Laemmli solution, and the proteins separated by electrophoresis. ...
