The transmissible spongiform encephalopathies of livestock.

ILAR Journal, 2015, Vol. 56, No. 1, 7–25 doi: 10.1093/ilar/ilv008 Article

The Transmissible Spongiform Encephalopathies of Justin J. Greenlee and M. Heather West Greenlee Justin J. Greenlee, DVM, PhD, Diplomate ACVP, is a research veterinary medical officer in the Virus and Prion Research Unit of the National Animal Disease Center, U.S. Department of Agriculture, Agricultural Research Service in Ames, Iowa. M. Heather West Greenlee, PhD, is an associate professor of biomedical sciences at the Iowa State University College of Veterinary Medicine. Address correspondence and reprint requests to Dr. Justin J. Greenlee, Virus and Prion Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, 1920 Dayton Avenue, Ames, IA 50010 or email [email protected].

Abstract 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 CreutzfeldtJakob 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. Key words: Bovine spongiform encephalopathy; cattle; goats; prion; protein misfolding; scrapie; sheep; transmissible spongiform encephalopathy

Prion Disease Prion diseases are a family of infectious protein-misfolding disorders resulting from aberrant folding and accumulation of the prion protein that leads to neurodegeneration (Aguzzi et al. 2008; Prusiner 1991). The misfolded form of the prion protein is commonly denoted PrPSc, as it was originally described as the disease causing agent of scrapie in sheep and goats (Prusiner et al. 1998). While normal cellular prion protein is in a predominantly alpha-helical structure (Prusiner 2001), misfolding of the prion protein results in a largely beta-sheet conformation (Pan et al. 1993). Misfolded prion molecules (PrPSc) act as a template for the aberrant refolding of normal cellular prion protein (PrPC) (Caughey and Raymond 1991) (Figure 1). Molecules of PrPSc form aggregates, become resistant to cellular digestion, and can

accumulate in lymphoid and nervous tissues, particularly the central nervous system (CNS) (DeArmond et al. 1985). In addition to the resistance of PrPSc to cellular digestion (Caughey et al. 1990), it is also resistant to digestion in vitro by proteinase K (PK) (McKinley et al. 1983; Oesch et al. 1985). Prion diseases result in accumulation of PrPSc and spongiform change in the brain (Budka 2003; Jeffrey and Gonzalez 2004) (Figure 2). Prion diseases are transmissible from one susceptible host to another and are therefore referred to as transmissible spongiform encephalopathies (TSEs).

Strains of Prion Disease Although all are caused by misfolded prion protein, TSEs within a species exist in multiple strains that may exhibit biochemical

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prion protein (PrPSc) serves as a template and catalyst for conversion of the endogenous normal cellular prion protein (PrPC) to PrPSc, which accumulates and oligomerizes, eventually leading to spongiform degeneration. Initiation of disease may be due to exposure to PrPSc (e.g., ingestion of contaminated material), spontaneous conversion of endogenous normal PrPC to PrPSc (e.g., sporadic TSE), or a mutation in the prion protein (e.g., genetic TSE).

differences, as well as different disease phenotype and pathogenesis (Cobb and Surewicz 2009; Sigurdson et al. 2007). In a given host, both PrPC and PrPSc are composed of identical amino acid sequences, so strain properties are maintained through conformational differences in PrPSc (Bessen and Marsh 1994; Legname et al. 2005). Strains can be differentiated by: • differing clinical signs (Pattison and Millson 1961) • incubation periods and lesion profiles in mouse models (Fraser and Dickinson 1968, 1973) • cellular and neuroanatomical deposition patterns of PrPSc (Bruce et al. 1989; Gonzalez et al. 2002) • molecular profile on Western blot• electrophoretic mobility (Bessen and Marsh 1992) • reactivity to antibodies near the PK cleavage point (Langeveld et al. 2011) • glycoform ratios (Stack et al. 2002) The reaction of different strain isolates to conformational antibodies (Safar et al. 1998) and stability assays (Vrentas et al. 2013) further supports the hypothesis that strains arise from differences in protein structure (Peretz et al. 2002). Strain diversity may arise from the inherent conformational flexibility of the prion protein, the presence of prion protein gene (PRNP) polymorphisms within a host species, or interspecies transmission events (Morales et al. 2007). Both bovine spongiform encephalopathy (BSE) and scrapie have strains classified as either classical or atypical. The classical strains have molecular, transmission, and phenotype characteristics consistent with the disease endemic within a population; and the atypical strains have molecular and epidemiologic characteristics that allow them to be differentiated from the classical strains (Benestad et al. 2003; Biacabe et al. 2004). The transmissibility and pathogenesis of a given TSE are dependent upon multiple factors including the PRNP

genotype of the host animal, the strain of TSE, and the route of exposure.

Prion Diseases are Diseases of Protein Misfolding Prion diseases share a common mechanism with other proteinmisfolding disorders including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and Huntington’s disease, in which misfolded disease-associated proteins catalyze a refolding of normal cellular protein that accumulates and is associated with neural degeneration (Fernandez-Borges et al. 2013; Hetz et al. 2003; Prusiner 2013; Soto 2003; Soto and Satani 2011). However, prion diseases are distinct from these other disorders in that they are readily transmissible to susceptible individuals via biologically significant routes of exposure.

Prion Diseases and Livestock Besides having fatal consequences to infected animals, scrapie and BSE are particularly important because of economic consequences to the livestock industry and potential public health concerns (Williams 2002). Scrapie can spread within a flock and brings economic losses due to production loss, export loss, and increased cost for carcass disposal, which ranges from $10–$20 million USD annually. The national scrapie eradication program began in 1952, was revisited in 2001, and implemented new surveillance practices in 2002–2003 that have decreased the number of cull sheep that are scrapie positive at slaughter by 90% (U.S. Department of Agriculture 2015). A single case of classical BSE was diagnosed in the United States in 2003 in an animal imported from Canada (Richt et al. 2007). As a result, loss of beef exports and domestic consumption was estimated to cost U.S. producers $4.7 billion USD in 2004. Since the first case of BSE was diagnosed,


Figure 1 Neurodegeneration in the transmissible spongiform encephalopathies is caused by the accumulation of misfolded prion protein. The infectious isoform of the

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of the dorsal motor nucleus of the vagus nerve in a sheep with clinical scrapie (B) compared with that in a negative control sheep (A) (hematoxylin and eosin stain). Immunohistochemistry to identify PrPSc in the same region reveals widespread immunoreactivity in sheep with scrapie (red reaction product; D) compared with no detectable signal in the control animal (C). Scale bars = 100 µm.

three additional atypical cases have been reported (Richt and Hall 2008; Richt et al. 2007; U.S. Department of Agriculture Animal and Plant Health Inspection Service 2012). The announcement of a single case of BSE in Canada in 2003 cost over $5.7 billion in total outputs (Fréchette 2005). A decade after the first reported case, beef exports from Canada were still approximately $1 billion less than they were in 2002. In the United Kingdom, where over 180,000 cattle were diagnosed with BSE and up to 3 million were likely to have been affected (Donnelly et al. 2002), the cost to the public was more than £5 billion. Loss of exports, consumer safeguards, compensation payments, and decrease in consumer demand all contribute to the costs incurred when additional cases of BSE are diagnosed.

Prion Disease and Human Health There are a number of TSEs that have been described in humans. They can be categorized as sporadic (unknown etiology), genetic, or acquired (Sikorska and Liberski 2012). The acquired TSEs in humans include iatrogenic Creutzfeldt-Jakob disease (iCJD), Kuru, and variant CJD (vCJD). Kuru and iCJD are the result of transmission between humans (Will 2003), and vCJD is a zoonotic disease

in which BSE from cattle infects humans that have ingested contaminated beef (Dormont 2002).

TSEs of Livestock Experimentally, TSEs of livestock are of interest for several reasons. Firstly, interspecies transmission experiments are crucial for the food animal industry to ensure the safety of the food supply. Secondly, TSEs in ruminants provide a well-characterized large animal model of protein-misfolding neurodegenerative disorders. Here we will review scrapie in sheep and goats, and BSE in cattle, experiments to assess interspecies transmission, approaches to antemortem diagnosis of TSEs in sheep and cattle, and finally the relevance of scrapie and BSE to human health and disease.

Scrapie Genetics of Susceptibility The TSEs are diseases of misfolded prion protein, and disease susceptibility is modulated by the amino acid sequence of the host’s prion protein. The codons of PRNP that are especially


Figure 2 Spongiform change and accumulation of PrPSc in the brain stem at the level of the obex. Spongiform change is marked within the neuropil and neuronal perikarya

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Classical Scrapie in Sheep Scrapie is the first described and most studied transmissible spongiform encephalopathy. Since the first description nearly 300 years ago in the United Kingdom and other western European countries, scrapie has been reported throughout the world with the notable exception of Australia and New Zealand. The transmissibility of scrapie was demonstrated by Cuillé and Chelle in 1936 (Schneider et al. 2008). It most likely spread through the export of subclinically affected animals (Detwiler and Baylis 2003). The first case of scrapie in the United States was reported in 1947 in imported sheep. This naturally occurring disease has a low

incidence in affected flocks and now is rare due to eradication programs. Transmission of scrapie from suspensions of brain and spinal cord from infected animals by intraocular, epidural, subcutaneous, and intracranial routes was demonstrated as early as 1936 (Schneider et al. 2008). Later, the accidental inclusion of tissues from a scrapie-infected flock in a louping-ill vaccine demonstrated that brain, spinal cord, and spleen contained infectious material; the agent was resistant to formalin; subcutaneous injection was sufficient for transmission; and the incubation period was 2 years or more (Gordon 1946). Natural transmission within flocks is thought to occur most commonly from the ewe to offspring of susceptible genotypes through the placenta and placental fluids, resulting in vertical transmission to susceptible offspring and the potential for horizontal exposure at the time of lambing (Touzeau et al. 2006). The demonstration of PrPSc in the placenta has been shown to be associated with placentomes of genetically susceptible offspring (Andreoletti et al. 2002) and in small amounts in the cotyledons of lambs of resistant genotypes that share the same uterine horn with a sibling of a susceptible genotype (Alverson et al. 2006). In addition to the presence of PrPSc in the cotyledons, infectivity of fetal tissues supports the possibility of in utero transmission (Spiropoulos et al. 2014). Transmission by milk and colostrum represent an additional risk of transmission to susceptible offspring born to infected dams (Konold et al. 2008, 2013). Environmental contamination leading to oral exposure of the scrapie agent is a major route of entry (Andreoletti et al. 2000; Ersdal et al. 2005; Gough and Maddison 2010; Ryder et al. 2004; van Keulen et al. 2002). Testing of infectivity of semen from scrapie-infected rams suggests that artificial insemination or natural breeding represents a neglible risk for scrapie transmission (Sarradin et al. 2008). However, potential prion seeding activity has been demonstrated in semen from infected rams by in vitro assays, suggesting that this route of transmission may require further study (Rubenstein et al. 2012). There is a long asymptomatic period lasting 2 to 7 years (depending on dose and genotype of sheep) between initial exposure to scrapie and the development of clinical signs. After ingestion, PrPSc crosses the intact intestinal barrier at the level of the enterocytes and passes rapidly into lymph and blood. These initial PrPSc steps are identical in susceptible and resistant sheep; however, only in susceptible sheep has PrPSc accumulation been shown to subsequently take place in lymphoid structures ( particularly in association with follicular dendritic cells) (Heggebo et al. 2000; Jeffrey et al. 2006). The first replication of the prion agent is within the lymphoreticular system, most likely the ileal Peyer’s patches (Andreoletti et al. 2000; Jeffrey et al. 2006). Early in the course of disease, PrPSc can be demonstrated in tonsil, spleen, and retropharyngeal and mesenteric lymph nodes (Muramatsu et al. 1994; Race et al. 1992). Regardless of the first site of replication, the abnormal prion spreads throughout lymphoid organs and is detectable for months prior to being detectable in the brain. Abnormal prion is detectable by immunohistochemistry in the lymphoid follicles, which allows for potential antemortem diagnosis using biopsies from the third eyelid (O’Rourke et al. 2000), tonsil (Schreuder et al. 1998), or rectal mucosa (Gonzalez, Horton et al. 2008). Immunoreactivity is most apparent at the center of the follicles and has been localized to both follicular dendritic cells and tingible body macrophages (Jeffrey et al. 2000). Oral uptake requires normal numbers of Peyer’s patches, but not enteric lymphocytes (Prinz et al. 2003), suggesting an important role of follicular dendritic cells (Mabbott and Bruce 2002). One factor in the greater relative susceptibility of younger sheep as compared with older sheep may have to do with more


significant in determining susceptibility or resistance to scrapie in sheep include 136, 154, and 171. PRNP haplotypes correlated with increased susceptibility are 136 valine (V), 154 arginine (R), and 171 glutamine (Q) (VRQ), whereas 136 alanine (A) (Goldmann et al. 1990; Hunter et al. 1991), 154 histidine (H) (Laplanche et al. 1993), and 171 arginine (R) (AHR) (Goldmann et al. 1994; O’Rourke et al. 1997) are associated with resistance to natural scrapie. Codon 171 appears to have the most discernible influence, where sheep with 171QQ are susceptible and 171RR are resistant. Another amino acid, lysine (K), occurs at codon 171 in some breeds of sheep. Similarities in charge and structure suggest that K may behave similarly to R. Sheep with a single 171K allele have prolonged incubation times after intracranial inoculation (Greenlee, Zhang et al. 2012), but it has not been determined whether sheep homozygous for the 171K allele will be resistant to scrapie or have prolonged incubation times. A significant reduction in the number of scrapie cases has been achieved in many locations by instituting breeding programs to increase the ARR allele in the sheep population in conjunction with removing affected animals. Polymorphisms that occur with lesser frequency, such as a substitution of methionine (M) by threonine (T) at codon 112 (Gonzalez et al. 2012; Laegreid et al. 2008) or leucine (L) by phenylalanine (F) at codon 141(Gonzalez et al. 2012), also have been shown to increase resistance to scrapie. Allelic variation in PRNP has been shown to modulate incubation times and susceptibility for scrapie in sheep. Scrapie in goats has been less well studied than in sheep, but numerous (at least 37) polymorphisms have been described in goat breeds worldwide (Acin et al. 2013). A subset of these potential amino acid substitutions appear to affect scrapie susceptibility: G127S (Goldmann et al. 2011), I142M (Goldmann et al. 1996), N146S/D (Papasavva-Stylianou et al. 2007), H154R (Barillet et al. 2009; Billinis et al. 2002; Papasavva-Stylianou et al. 2011; Vaccari et al. 2006), Q211R (Barillet et al. 2009), and Q222K (Acutis et al. 2006; Barillet et al. 2009; Vaccari et al. 2006). Experimental studies suggest that M142 prolongs incubation period (Goldmann et al. 1996) but does not result in resistance to scrapie (Lacroux, Perrin-Chauveneau et al. 2014). However, H154, Q211, and K222 carriers are all resistant to scrapie after oral exposure (Lacroux, Perrin-Chauveneau et al. 2014). Cases have been reported in heterozygous K222 animals, but not in homozygous K222 (Barillet et al. 2009; Corbiere et al. 2013). The fact that a small number of K222 goats developed scrapie after intracranial inoculation confirms that resistance to scrapie is not absolute. Goats have been naturally infected with both atypical scrapie (Le Dur et al. 2005; Seuberlich 2007) and BSE (Eloit et al. 2005). Similar to sheep, the risk of developing atypical scrapie has been shown to be higher in H154 goats (Colussi et al. 2008). Results of studies using transgenic mice expressing K222 demonstrated resistance to bovine spongiform encephalopathy, further suggesting K222 would make a good candidate for selective breeding programs to enhance scrapie resistance in goats (Aguilar-Calvo et al. 2014).


prominent gut-associated lymphoid tissues in young animals (Press et al. 2004). Because of widespread lymphoid accumulation of PrPSc during the asymptomatic phase, infected sheep may serve as a source of prion contamination to the environment long before the onset of clinical signs. Despite the participation of the lymphoid system in the pathogenesis of scrapie, there is no specific humoral immune response (Porter et al. 1973) due to tolerance to host PrPC (Bendheim et al. 1992), which has the same amino acid sequence.

Atypical Scrapie

demonstrated in tissues outside the CNS (ileum and spleen [Simmons et al. 2011]); skeletal muscle, lymphoid tissues, peripheral nerves [Andreoletti et al. 2011]) of affected individuals despite a failure to detect PrPSc (Simmons et al. 2011). Evidence of infectivity outside of the CNS suggests that there is the possibility for natural transmission or spread through contaminated animal feed. Atypical scrapie has also been reported in goats (Seuberlich 2007), where the molecular profile on western blot is similar to atypical scrapie in sheep, but the distribution of lesions within the brain is more rostral (thalamus and midbrain) than that of atypical scrapie of sheep (Seuberlich 2007). Classical Scrapie in Goats Scrapie in goats was first described in 1939 after experimental transmission from sheep, and the first natural case was described shortly thereafter (Fast and Groschup 2013). Goats with scrapie are commonly in mixed herds with sheep, but scrapie can also occur in goats that have not had contact with sheep (Wood et al. 1992). Similar to what is observed in sheep with scrapie, assessment of goats with natural scrapie suggests widespread involvement of the lymphoreticular system, specifically retropharyngeal lymph node and palatine tonsil (Gonzalez et al. 2009). In contrast to what is commonly observed in sheep, accumulation of PrPSc in the brain of goats with scrapie appears to be highly dependent upon genotype at codon 142. In a study of naturally infected goats, 75% of M142 carriers had PrPSc in the brain as opposed to only 35% of isoleucine homozygotes (II142) (Gonzalez et al. 2010). Accumulation of PrPSc in placentas from goats with scrapie has been reported to be relatively lower when compared with what has been observed in the placentas of scrapie-positive sheep (O’Rourke et al. 2011). Atypical Scrapie in Goats Atypical scrapie also has been described in goats (Le Dur et al. 2005; Seuberlich 2007). Similar to sheep, a histidine substitution at 154 is a risk factor for atypical scrapie in goats (Colussi et al. 2008), and PrPSc has not been demonstrated in the lymphoid tissues of affected animals (Seuberlich 2007).

Bovine Spongiform Enchephalopathy What is now known as classical bovine spongiform encephalopathy (BSE; mad cow disease) was first diagnosed in the United Kingdom in 1985 (Wells et al. 1987). Cattle with BSE are symptomatic with neurologic signs of incoordination and weight loss, and they demonstrate unusual aggression in some cases (Wilesmith 1988). The origin of BSE remains unknown. Early theories suggested that BSE was the result of passage of a scrapie- like disease into the cattle population (Wilesmith et al. 1988), potentially via the use of ruminant-derived meat and bone meal (MBM) as a food source. However, studies conducted in the United States and United Kingdom have shown that scrapie does not transmit to cattle by oral routes of inoculation (Cutlip et al. 2001) and, when it does transmit by intracranial inoculation, the resultant disease is distinguishable from BSE by clinical signs, molecular profile of PrPSc, and PrPSc deposition patterns in brain sections (Cutlip et al. 1994, 1997; Konold et al. 2006). Preliminary evidence from laboratory animal models suggests that, after multiple passages, Htype atypical BSE (defined below), of presumed sporadic origin, can assume a classical BSE phenotype (Bencsik et al. 2013). Whatever the initial infectious source, the widespread BSE outbreak in the United Kingdom was due, in large part, to the feeding of MBM to cattle. MBM contains central nervous system tissues and other tissues that are known to contain the abnormal prion protein.


Atypical (Nor98) scrapie is a second TSE of sheep that was first described in Norway in 1998 (Benestad et al. 2003, 2008). These cases were defined as atypical based on several criteria: clinical presentation, molecular characteristics of the abnormal prion protein, distribution of PrPSc within infected sheep, genotypes of affected sheep, and epidemiology. Since 1998, additional cases have been diagnosed throughout Europe (Arsac et al. 2007; Dagleish et al. 2008; De Bosschere et al. 2007; Fediaevsky et al. 2008; GavierWiden 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. Cases of atypical scrapie are distinguished by a western blot profile that has a 5-band profile with a prominent lower band at approximately 12 kDa (or lower depending on conditions [Klingeborn et al. 2006]), whereas classical scrapie has the nonglycosylated band at approximately 19–21 kDa (Hayashi et al. 2005; Hope et al. 1999; Somerville and Ritchie 1990). Furthermore, the PrPSc molecule of atypical scrapie is relatively sensitive to proteinases (Klingeborn et al. 2006), resulting in discrepant diagnostic test results depending on the test method used (Baron, Biacabe et al. 2007; Buschmann et al. 2004). The spongiform change and PrPSc deposition in atypical scrapie cases occur predominantly in the cortices of the cerebellum and the cerebrum, rather than the medulla oblongata as seen in classical sheep scrapie. This does not fit the model of oral infection of intestinal Peyer’s patches followed by ascension through the vagus nerve to the brainstem. The agent has not been described outside of the CNS: currently available methods have failed to demonstrate PrPSc in the lymphoid tissues of sheep affected with atypical scrapie (Andreoletti et al. 2011; Benestad et al. 2003). Atypical scrapie cases are sometimes identified in sheep with genotypes considered resistant to classical scrapie: affected sheep in the original report carried at least one A136H154Q171 allele (Benestad et al. 2003), and subsequent studies have demonstrated an increased risk of atypical scrapie in sheep with the AHQ and AF141RQ haplotypes (Saunders et al. 2006).. Sheep with the ARR haplotype do not appear to be protected against developing atypical scrapie (De Bosschere et al. 2007; Saunders et al. 2006). Because atypical scrapie is usually detected in only one sheep in a flock, does not appear to be contagious or transmits very poorly under natural conditions (Fediaevsky et al. 2010), and is usually found in older animals, a sporadic etiology (i.e., spontaneous occurrence or de novo pathogenesis) has been suggested. Supporting the concept of a sporadic occurrence is the long-held categorization of certain forms of CJD having a sporadic etiology and recent evidence that abnormally-folded prions can arise de novo in experimental systems. Interestingly, atypical scrapie has been transmitted experimentally to AHQ sheep by the intracranial (Simmons et al. 2007) and oral (Simmons et al. 2011) routes, and infectivity has been

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Classical BSE Classical BSE was the first cattle TSE to be recognized (Wells et al. 1987). Classical BSE is acquired by consumption of contaminated foodstuffs. Epidemiological studies suggest dietary supplements, in particular MBM containing CNS tissues with PrPSc, as the source of the outbreak in the United Kingdom (Wells et al. 1991). Studies in Great Britain have not detected vertical (Wilesmith et al. 1997) or horizontal transmission of classical BSE between cattle (including an absence of evidence for transmission from “contaminated” pastures) (Kimberlin 1992). Clinical signs of BSE include changes in temperament, abnormal responses to environmental stimuli, abnormal posture, incoordination and difficulty in rising, decreased milk production, and loss of body weight despite continued appetite (Saegerman et al. 2004). It is not possible to know the precise time of exposure in natural cases, but adult cattle are at relatively lower risk of developing classical BSE: most clinical cases in Great Britain occurred in dairy cows between 3 and 6 years of age, with the highest apparent susceptibility in the first 6 months of life (Arnold and Wilesmith 2004). Atypical BSE in Cattle Several hypotheses have been proposed to explain the etiology of atypical BSE cases. At the forefront of these is the possibility that both H-type and L-type BSE may arise spontaneously in cattle. This is drawn from atypical BSE’s parallels to sporadic CJD in humans, specifically, it’s prevalence in older hosts and a comparably low incidence (Tranulis et al. 2011). The fact that the number of cattle affected by classical BSE has fallen precipitously in response to control measures, while the number of cattle affected by atypical BSE remains relatively stable over time, supports the hypothesis that these atypical cases occur spontaneously (Brown et al. 2006). Furthermore, prion gene promoter region polymorphisms that influence susceptibility to classical BSE do not appear to influence atypical BSE (Brunelle et al. 2007), and atypical BSE occurs as isolated cases in contrast to the clustering of cases observed for feedborne classical BSE. Finally, atypical BSE cases have occurred predominantly in cattle greater than 10 years of age, whereas classical cases have occurred in much younger animals (Biacabe et al. 2004; Casalone et al. 2004). H-type BSE has been described in cattle from France (Biacabe et al. 2004), Germany (Buschmann et al. 2006), Japan (Sugiura et al. 2009), the Netherlands (Biacabe et al. 2007), Poland (Biacabe

Figure 3 Strains of BSE can be differentiated by molecular profile. Western blot after proteinase-K digestion reveals three bands representing (from higher to lower) the diglycosylated, monoglycosylated, and nonglycosylated forms of the prion protein. Low-type BSE (BSE-L) in lane 1 is the lowest molecular weight compared with high-type BSE (BSE-H, lane 2) or classical BSE, which has an intermediate molecular weight (lane 3). There is no detectable PrPSc in the brain of the negative control animal (lane 4). The different molecular weights of BSE-L, BSE-H and classical BSE are due to differential cleavage of the n-terminal region of the abnormal prion protein.

et al. 2007), Switzerland (Tester et al. 2009), the United Kingdom (Sohn et al. 2009), and the United States (Richt et al. 2007). The molecular phenotype of H-BSE cases on western blot (Figure 3) is characterized by (1) a higher molecular mass of the unglycosylated PrPSc isoform; (2) a strong labeling of all 3 PrPSc polypeptides (unglycosylated, monoglycosylated, and diglycosylated isoforms) with the PrP-specific monoclonal antibodies that bind to the N-terminus (such as mAb p4) (Biacabe et al. 2004); and (3) a second unglycosylated band at approximately 14 kDa when developed with antibodies that bind in the C-terminal region (amino acids 154–236), such as SAF 84 (Biacabe et al. 2007). Brains of animals inoculated with H-type BSE have similar spongiform lesions at the level of the obex, but have more vacuolation in the frontal cortex than occurs in the brains of cattle similarly challenged with classical BSE (Konold et al. 2012). Immunoreactivity for PrPSc in brains of cattle inoculated with BSE-H is distinct from that of classical BSE, as well as BSE-L, the other form of atypical BSE (Greenlee, Smith et al. 2012; Okada et al. 2011). L-type BSE has been found in cattle in Italy (Casalone et al. 2004), Japan (Yamakawa et al. 2003), Germany (Buschmann et al. 2006), Belgium (De Bosschere et al. 2004), and Canada


Prior to the identification of the epidemic, many cattle with BSE were being slaughtered, with offal entering MBM, thus facilitating distribution of infectious material (Wilesmith et al. 1991). Elimination of BSE focuses on preventing tissues with highest risk of causing infection (called specified risk materials or SRM) from getting into the human or animal food supply. In the United States, SRM includes brain, skull, spinal cord, trigeminal and dorsal root ganglia, eyes, and vertebral columns of cattle greater than 30 months of age, and tonsil and distal ileum of all cattle (U.S. Department of Agriculture 2014). Surveillance efforts are focused on cattle most likely to have BSE: dead, diseased, immobile, or disabled cattle with neurologic signs and those greater than 30 months of age (U.S. Department of Agriculture 2014). BSE can be subdivided into at least three strains: classical, Htype, and L-type. The H- and L- designations are based on higher or lower apparent molecular mass profiles of the unglycosylated PrPSc band on a western blot (Figure 3), and are collectively referred to as atypical BSE (Wells 2007). The vast majority of BSE cases are the classical subtype that was associated with the UK BSE epizootic.


Collinge 2004). To fully assess potential risks to animal and human health, it is important to study interspecies transmission of scrapie and BSE in addition to the other known animal TSEs: chronic wasting disease (CWD) and transmissible mink encephalopathy (TME). Furthermore, species barrier can be studied through the use of transgenic mice expressing the PRNP of the species of interest or in in vitro conversion assays (discussed below). Chronic Wasting Disease (CWD) Naturally occurring CWD has been documented in mule deer (Odocoileus hemionus hemionus), black-tailed deer (Odocoileus hemionus columbianus), white-tailed deer (Odocoileus virginianus), Rocky Mountain elk (Cervus elaphus nelsoni), and moose (Alces alces shirasi) (Baeten et al. 2007; Williams 2005). The disease was first recognized in a captive population of mule deer at the Colorado Division of Wildlife Foothills Wildlife Research Facility in Fort Collins in 1967, although identification of the disease as a TSE did not occur until 1978 (Williams and Young 1980). The origins of CWD are uncertain, but white-tailed deer appear to be readily susceptible to scrapie of North American origin (Greenlee et al. 2011). Transmissible Mink Encephalopathy (TME) Transmissible mink encephalopathy was identified in ranchraised mink (Neovison vison). It was first documented in Wisconsin, in 1947 (Eckroade et al. 1973), and the last reported outbreak in the United States was in 1985 (Marsh et al. 1991). TME is a foodborne disease that has been experimentally transmitted to a variety of animal species (Eckroade et al. 1970, 1973). The origin of TME is unknown, but has been associated with feeding downer cattle to farmed mink (Marsh and Hartsough 1988). Molecular similarities and results of rodent studies suggest a high degree of similarity between TME in cattle and BSE-L (Baron, Bencsik et al. 2007).

Experimental Interspecies Transmission Prion diseases can be transmitted from one species to another. Experimental cross-species transmission of TSE agents provides valuable information about potential host ranges and differentiation from known TSEs and may lead to clues about their origins. The most common routes of inoculation to test for interspecies transmission are oral and intracranial (IC). Oral inoculation is generally effective in intraspecies transmission studies, is considered a natural route by which animals acquire TSEs, and best mimics natural disease pathogenesis. Intracranial inoculation is very efficient for testing whether a new host has any potential to acquire a TSE from a given species since it requires smaller dosage of inoculum and results in higher attack rates and reduced incubation periods than does oral transmission. A species resistant to a TSE by IC inoculation would have negligible potential for successful oral transmission. Unsuccessful attempts at interspecies transmission led to the concept of a species barrier. The species barrier phenomenon limits transmission of prions between different mammalian species and is influenced by mismatches between host and recipient prion amino acid sequence and the resulting structures and folding (Beringue et al. 2008; Collinge and Clarke 2007; Hill and Collinge 2001, 2004; Moore et al. 2005; Scott et al. 1993; Vanik et al. 2004). The species barrier, even in closely related species, can manifest as complete lack of susceptibility, incomplete attack rates, or prolonged incubation times. Primary passage is generally not efficient between species, and sequential passages are required for a TSE strain to stabilize in a new species (Hill and

Experimental Transmission of Bovine and Cervid TSEs to Sheep and Goats Numerous experimental studies have been done to better understand the potential transmission of BSE, CWD, and TME to sheep and goats. The likelihood that sheep were exposed to the same contaminated feedstuffs as cattle during the UK BSE epizootic and detection of a natural case of BSE in a goat (Eloit et al. 2005) heightened concern about the potential transmission of BSE to small ruminants. Sheep are readily infected with the agent of BSE by the oral route, with the highest susceptibility prior to weaning (Hunter et al. 2012), and resulting in infectivity throughout lymphoid tissues (Foster et al. 1993; Foster, Parnham, Hunter et al. 2001). In most cases, sheep have PrPSc accumulation in tissues similar to those infected with scrapie, but some sheep develop neuroinvasion without accumulation of PrPSc in lymphoid tissues (Jeffrey, Ryder et al. 2001). Maternal and contact transmission of BSE to sheep appears to be quite inefficient (Foster et al. 2004), although natural transmission to offspring born to experimentally infected ewes can occur (Bellworthy et al. 2005). Strain-specific processing of misfolded PrP within the nervous system and lymphoid tissues leads to differences in immunohistochemical staining patterns depending on the antibodies used, which allows for differentiation of BSE from scrapie in sheep (Jeffrey, Martin et al. 2001). Similarly,western blotting can be used to differentiate BSE from scrapie in sheep by molecular weight, glycoform ratio, and different apparent N terminal cleavage point after PK digestion using either brain (Stack et al. 2002) or lymph


(Dudas et al. 2010). Western blot analysis demonstrates that the L-type form has a lower molecular mass of the unglycosylated PrPSc isoform when compared with classical BSE. In experimentally challenged animals, both BSE-H and BSE-L appear to have shorter incubation times than classical BSE (Balkema-Buschmann, Ziegler et al. 2011; Konold et al. 2012). Polymorphisms in the PRNP gene in cattle were thought to be rare (19 single nucleotide polymorphisms that are not associated with changes in the amino acid sequence) and had not been previously associated with susceptibility to BSE (Clawson et al. 2006). In 2006, however, a case of BSE that was diagnosed in the United States led to the identification of a new prion protein allele, K211 (Richt and Hall 2008), that is associated with H-type BSE and is heritable (Nicholson et al. 2008). This amino acid polymorphism is homologous to E200K (where lysine (K) replaces glutamate (E)) in humans, which is the most common hereditary form of Creutzfeldt-Jakob disease (Kovacs et al. 2005). The average onset of disease in individuals with the E200K mutation varies from the third to the seventh decade of life (Schelzke et al. 2012), implying that changes that occur during the aging process are linked to the progression of disease. It remains to be determined what mechanisms are associated with the onset of disease in E211K-associated H-type BSE, but further study of this disease could lead to breakthroughs in understanding the pathogenesis of the inherited TSEs of humans. In cattle, the BSE agent, unlike its counterpart scrapie in sheep and goats, amplifies primarily in the CNS and does not involve widespread accumulation in the lymphoreticular system (Balkema-Buschmann, Fast et al. 2011). In cattle with classical BSE, abnormal prion protein (PrPSc) has been consistently detected only in the tonsils and Peyer’s patches of the distal ileum (Espinosa et al. 2007; Terry et al. 2003; Wells et al. 2005), although there is evidence for more peripheral distribution at late stages of the disease (Balkema-Buschmann, Eiden et al. 2011).

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Experimental Transmission of Sheep and Cervid TSEs to Cattle Interspecies transmission of TSEs to cattle has been extensively studied (Hamir et al. 2011). During the 1990s, the possibility that U.S. strains of sheep scrapie might cause BSE in cattle was assessed experimentally. Intracranial inoculations resulted in 100% transmission of scrapie to cattle between 14 and 18 months following inoculation (Cutlip et al. 1994). A separate study using multiple simultaneous routes of inoculation (including IC) found only 20%–40% transmission of scrapie to cattle depending on the source of inoculum and a longer incubation period of 24 to 48 months prior to affected cattle exhibiting anorexia, weight loss, leg and back stiffness, incoordination, and rear leg weakness, eventually leading to severe lethargy and ataxia (Clark et al. 1995). Following oral exposure to the scrapie agent, cattle did not develop symptoms of neurological disease, spongiform lesions, or PrPSc deposits in the CNS by the time of examination 8 years after inoculation (Cutlip et al. 2001). In contrast, oral inoculation of the agent of BSE into cattle is a highly efficient means of transmission (Wells et al. 2007). Scrapie in cattle can be differentiated from BSE microscopically: while there is no evidence of spongiform changes in the brains of scrapie-affected cattle, spongiform changes are usually observed in clinical BSE cases (Scott et al. 1990). Further, immunoreactivity for PrPSc in scrapie-affected cattle was observed predominantly in neuronal cell bodies with relatively little accumulation in the neuropil (Cutlip et al. 1994, 1997), in contrast to BSE where there is a diffuse distribution of PrPSc in the CNS (Scott et al. 1990). Despite the proposed linkage of the BSE epidemic to feed contamination by a scrapie-

like agent (Wilesmith et al. 1988), scrapie isolates have only been transmitted to cattle by IC inoculation, and the pathology and clinical disease differed from both BSE in cattle and scrapie in sheep (Cutlip et al. 1994, 1997; Konold et al. 2006). Therefore, current experimental evidence from scrapie transmission studies into cattle does not support the hypothesis that the U.K. BSE epidemic originated from the feeding of scrapie PrPSc to cattle, however, only a limited number of scrapie isolates have been thoroughly tested in cattle. The recognition of CWD (Williams 2002) in captive and freeranging cervids in the United States raised questions about the possible transmissibility to other ruminant species that share the same pastures or range. Studies have been done to investigate the potential of cattle to serve as a host to the agent of CWD derived from mule deer, white-tailed deer, and elk sources. IC inoculation of cattle with the CWD agent from mule deer resulted in accumulation of PrPSc in the CNS, indicating that amplification of the abnormal CWD prion had occurred, although there was only subtle spongiform change in the brain (Hamir et al. 2001). The initial study demonstrated a low (38%) attack rate of mule deer CWD upon first passage in cattle (Hamir et al. 2005), but a second IC passage of mule deer CWD transmitted the disease to all inoculated cattle within 16.5 months after inoculation (Hamir, Kunkle, Miller, Greenlee et al. 2006). IC inoculation of the agent of CWD from white-tailed deer into cattle showed that the white-tailed deer inoculum had a higher attack rate (86%) in cattle than the mule deer CWD inoculum used previously, however, spongiform change in the brain was not observed (Hamir et al. 2007). None of the cattle given the same inoculum orally (50 g of pooled brain/animal) has shown any evidence of prion disease up to 9 years after inoculation (Williams 2005), however, calves were not inoculated until 4 months of age. IC inoculation of cattle with the CWD agent from elk also resulted in a low rate of transmission: clinical signs ( poor appetite, weight loss, circling, and bruxism) occurred in 2 out of 16 cattle at 16 and 17 months post-inoculation with PrPSc, but no spongiform lesions were detected in the CNS (Greenlee, Nicholson et al. 2012). Regardless of CWD inoculum source, deposition of PrPSc appears to be similar and clearly distinguishable from BSE or scrapie in cattle: immunoreactivity is prominent in midbrain, brainstem, and hippocampus, with lesser immunoreactivity in the cervical spinal cord that occurs in multifocal and distinct aggregates confined to glial cells and associated neuropil. Additional studies are required to fully assess the potential for cattle to develop CWD through a more natural route of exposure, but low attack rates after IC inoculation and failure of transmission following oral exposure suggests that the risk of transmission through routes other than IC is low. Inoculation of cattle with the agent of TME from three different sources led to clinical disease and severe spongiform encephalopathy (Robinson et al. 1995), confirming earlier reports of TME transmission to cattle (Marsh et al. 1991). Subsequent IC inoculation of cattle with first and second passage cattle-adapted TME confirmed the earlier findings and also described for the first time the immunohistochemical and western blot characteristics (lower molecular weight of cattle-adapted TME vs. C-type BSE by western blot) of the accumulated PrPSc, which is dissimilar from experimental scrapie and experimental CWD in cattle (Hamir, Kunkle, Miller, Bartz et al. 2006). Studies conducted in ovinized transgenic mice demonstrated that cattle-passaged TME presented with the same phenotypic characteristics as atypical Ltype BSE (Baron, Bencsik et al. 2007). However, experimental studies investigating the oral route of transmission of TME to cattle have not been reported.


node samples (Langeveld et al. 2006). The incubation period of BSE in sheep is genotype dependent and, in contrast to sheep with scrapie, ARQ/ARQ and AHQ/AHQ sheep have the shortest incubation times (Foster, Parnham, Chong et al. 2001), with a V136 allele greatly increasing the incubation time (Goldmann et al. 1994). Sheep carrying a R171 allele are not entirely resistant to BSE, but do have prolonged incubation periods (Foster, Parnham, Chong et al. 2001). Only the M to T substitution at codon 112 has been shown to protect against challenge with BSE (Saunders et al. 2009). Attempts to transmit the agent of CWD from mule deer to sheep by intracranial inoculation resulted in PrPSc accumulation in only two of eight inoculated lambs (Hamir, Kunkle, Cutlip et al. 2006). PrPSc was observed in the brain and tonsil of one ARQ/ARQ sheep euthanized at the end of the 72-month study without developing clinical signs. One ARQ/VRQ sheep developed clinical signs and was euthanized at 35 months post-inoculation with extensive spongiform lesions throughout the brain, and PrPSc was demonstrated throughout the nervous and lymphoid systems, suggesting that sheep with the V136 codon should be further investigated in their potential susceptibility to CWD (Hamir, Kunkle, Cutlip et al. 2006). Transmission of the agent of transmissible mink encephalopathy to sheep and goats by intracranial inoculation has been documented (Hadlow et al. 1987; Marsh et al. 1969; Zlotnik and Barlow 1967). Incubation times ranged from 45 to 80 months in sheep and 31 to 40 months in goats (Hadlow et al. 1987). A mink bioassay (sheep tissues intracranially inoculated into a mink host) was used to demonstrate infectivity in lymph node and spleen material (Marsh et al. 1969). These studies were conducted prior to the ability to determine the PRNP genotype of sheep and goats, so no information on the effect of genotype on susceptibility or resistance is available for TME.


Transgenic mice that have been engineered to express either the bovine PRNP sequence (Buschmann and Groschup 2005; Castilla et al. 2003) or ovine PRNP sequences (Kupfer et al. 2007; Vilotte et al. 2001) have greatly enriched interspecies transmission studies of scrapie and BSE. While a thorough discussion of the body of work using transgenic mice is outside the scope of this review, it is worth making note that transgenic mice have been a useful tool in understanding the pathogenesis of scrapie and BSE. Examples include the use of ovinized mice to characterize atypical scrapie (Griffiths et al. 2010) or to resolve scrapie cases with a BSE-like molecular profile (Beck et al. 2012) and the use of bovinized mice to assess potential infectivity of tissues from animals with BSE (Fast et al. 2013) or the potential of multiple passages of atypical BSE to promote conversion to a classical BSE phenotype (Torres et al. 2011).

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TSEs of Livestock And Human Health Our understanding of the risks of animal TSEs to human health comes from several lines of evidence. In addition to epidemiological studies, the potential for an animal TSE to be transmitted to humans has been experimentally evaluated in two different ways. The first is via the experimental use of “humanized: transgenic mice, where the mouse prion protein gene (PRNP) has been knocked out, and the mice instead express the human PRNP gene (Kitamoto et al. 1996). The second approach that has been used to evaluate the potential of an animal prion disease to affect humans is the in vitro conversion assay protein-misfolding cyclic amplification (PMCA, see below), where human brain homogenate is used as a substrate (Jones et al. 2007; Levavasseur et al. 2014).

Scrapie Considerable evidence suggests that scrapie does not pose a risk to human health. Despite the presence of scrapie throughout much of the world for nearly 300 years, there is no evidence that scrapie has ever been transmitted to humans. In former times, it was a widespread management practice to slaughter sheep with clinical signs for human consumption (Schneider et al. 2008), but no increase in neurologic disease has been associated with consuming products from sheep. A number of studies have demonstrated that transgenic mice expressing the human prion are not susceptible to classical (Gombojav et al. 2003) or atypical scrapie (Wadsworth et al. 2013; Wilson et al. 2012). Further, using human brain or mouse brain expressing human PRNP as substrate in PMCA conversion assays fails to demonstrate replication of scrapie prions (Jones et al. 2007). However, recent results demonstrated transmission of classical scrapie strains to mice overexpressing the human prion protein, resulting in a molecular phenotype with similarities to human sporadic Creutzfeld-Jakob disease (Cassard et al. 2014), indicating the need for continued research in this area to clarify any potential risks of scrapie to human health.

Bovine Spongiform Encephalopathy Unlike scrapie, BSE is a known risk to human health. Due to conservation of molecular phenotype upon transmission of BSE to multiple species including mice expressing the human prion protein it is accepted that variant Creutzfeldt-Jakob disease (vCJD), which emerged in the United Kingdom in the mid-1990s, is caused by the ingestion of contaminated beef from cattle with BSE (Bruce et al. 1997; Collinge et al. 1996; Hill et al. 1997; Will et al. 1996). In vitro conversion assays demonstrate that BSE

Diagnosis of TSEs in Livestock Definitive diagnosis of prion disease depends upon detection of misfolded prion protein (PrPSc) in the diseased animal. There is a significant effort to develop antemortem tissue collection and detection techniques; however, the current gold standard for diagnosis is the postmortem detection of PrPSc in brain tissue (usually brain stem at the level of the obex) by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, or western blot (NCBA 2014). Current diagnostics that are based on detection of PrPSc perform well on clinically affected animals for all described strains of BSE (Gray et al. 2012). The major diagnostic challenge is detecting infected animals in the preclinical stage. Emphasis for the past decade has been on the development and optimization of techniques to amplify misfolded PrPSc in a diagnostic sample so that it can be detected (Orru et al. 2012). Two basic approaches include PMCA (Saa et al. 2006), which uses brain homogenate as a substrate that is “seeded” by the diagnostic sample, and Quaking-Induced Conversion (QuIC), which uses recombinant protein as the substrate to be seeded by misfolded protein in a diagnostic sample (Atarashi et al. 2008). PMCA and QuIC have been used to detect PrPSc in biological samples including blood (Lacroux, Comoy et al. 2014), urine (Moda et al. 2014), and saliva (Okada et al. 2012).

Scrapie Sheep and goats infected with scrapie accumulate PrPSc in the lymphoreticular system allowing the opportunity for antemortem sampling and testing by immunohistochemistry (Figure 4) or ELISA. Biopsy of rectal mucosa (Gonzalez, Dagleish et al. 2008), tonsil (Schreuder et al. 1998), or third eyelid (O’Rourke et al. 2000) holds promise for antemortem detection of infected animals (Dennis et al. 2009; Monleon et al. 2011). The sensitivity of immunohistochemistry to detect PrPSc in rectal mucosal biopsies from field samples of preclinically affected sheep has been reported to be as high as 89.4% (Dennis et al. 2009), although others report a more modest result of 48% (Monleon et al. 2011). A study of goats with natural scrapie reported that only 42% of preclinical scrapie positive goats had positive rectal mucosal biopsies (Gonzalez et al. 2009), suggesting that rectal biopsy of goats may be of lower sensitivity than for sheep.

Bovine Spongiform Encephalopathy There is much less accumulation of PrPSc in the periphery of cattle infected with BSE when compared with sheep and goats with


can convert PrPC to PrPSc in human substrate much more efficiently than other animal TSEs (Barria et al. 2014). Susceptibility to vCJD is greatly affected by a polymorphism at codon 129 in the human PRNP, which can be either methionine or valine (Collinge et al. 1991). All diagnosed cases of vCJD have been homozygous MM at codon 129 (Saba and Booth 2013). Experiments in humanized mice further support the susceptibility conferred by this polymorphism (Wadsworth et al. 2004). Quite interestingly, a recent large-scale survey of human appendices removed in the United Kingdom identified abnormal PrPSc in 16 of 32,441 samples. The genotypes of the positive specimens were a high proportion VV at codon 129 (Gill et al. 2013) raising the possibility that the 129 genotype affects incubation time, rather than absolute susceptibility. Classical foodborne BSE appears to be the primary risk to human health, as atypical forms of BSE are not transmissible to humanized mice (Kong et al. 2008; Wilson et al. 2012).

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scrapie. Thus, techniques for early detection of scrapie-infected sheep and goats (i.e., rectal biopsy) cannot be applied to preclinical detection of cattle infected with BSE. In studies of cattle orally challenged with BSE, immunoreactivity for PrPSc has been reported in the ileal Peyer’s patches 4 to 6 months post challenge (Terry et al. 2003) and in the palatine tonsil starting at 10 months after the challenge (Wells et al. 2005). This is much earlier than the first detection of PrPSc immunoreactivity in the brainstem at 27 months post challenge (Espinosa et al. 2007). During the clinical phase of BSE in cattle, however, there is a centrifugal spread of abnormal prions to the periphery (Buschmann and Groschup 2005; Franz et al. 2012), which suggests peripheral tissues may be useful for diagnosis of clinically ill animals. Various studies have used samples from cattle experimentally challenged with BSE to further define the tissue distribution of prions in infected cattle (Bannach et al. 2013; Murayama et al. 2010). However, there is not, as yet, a validated antemortem test that can be used to screen cattle for BSE. A careful behavioral assessment of experimentally inoculated cattle demonstrates that numerous nonspecific behavioral changes can be observed prior to the clinical phase of BSE. Difficulty rising is the most consistent feature of BSE-challenged animals (Konold et al. 2012). While often observed months prior to the end stage of the disease, this behavioral feature did not have a consistent time course or onset (Konold et al. 2012). Our group has described changes in retinal function and morphology in cattle experimentally inoculated with TSEs (Greenlee, Smith et al. 2012; Smith and Greenlee 2014; Smith et al. 2008; Smith, Greenlee, Hamir, Greenlee 2009; Smith, Greenlee, Hamir, Richt et al. 2009). The retina is a part of the CNS, and accumulates PrPSc in experimentally and naturally occurring cases of prion disease in sheep and cattle (Greenlee et al. 2006; Hortells et al. 2006; Okada et al. 2011; Smith, Greenlee, Hamir, Richt et al. 2009). Using electroretinography (ERG) and optical coherence tomography (OCT) (Figure 5) we have demonstrated that changes in retinal function and retinal thickness are detectable from 5 to 19 months prior to BSE-inoculated cattle displaying clinical signs of disease (West Greenlee et al. 2015). To understand the impact that detection of PrPSc accumulation may have on public health, Arnold and colleagues estimated how the time course of PrPSc detection in cattle related to the incubation period in cattle experimentally challenged with BSE.

Based on their data, they estimate that, in cattle orally challenged with 100 g of infectious material, PrPSc accumulation is detectable in the brainstem 9.6 months prior to clinical disease. In cattle orally challenged with 1 g of infectious material, however, it was estimated that PrPSc would be detectable in the brain stem 1.7 months prior to the clinical onset of disease (Arnold et al. 2007).

Animal Models of TSEs The TSEs are a family of fatal, infectious neurodegenerative human and animal diseases that share a common mechanism of misfolding of the prion protein. The strong similarities between human and animal prion diseases offer advantages to biomedical research. Whereas human prion diseases are often diagnosed late in the course of disease after the onset of clinical signs and beyond the point of any possibility of productive intervention, prion diseases in animals have defined incubation periods, progression of lesions, and genotype susceptibilities that can be used to design prospective studies that may lead to methods of prevention or cure for prion diseases. There is a high likelihood that results of animal studies will translate to human medicine through improved diagnostics, prevention strategies, and policies to improve public health. Research to understand the animal prion diseases and the potential for interspecies transmission has lead to major policy changes to protect public health. Understanding the pathogenesis of BSE, including the tissue distribution of BSE prions, is critical to identifying SRM, which allows for removal of those tissues from the human and animal food supplies. Interspecies transmission studies in ruminants or using ruminant-derived prions has helped identify prion isolates with zoonotic risk and others that deserve further study to fully understand the potential zoonotic risk (Beringue et al. 2008; Hamir et al. 2011). Ruminants have been used extensively to study the transmission and differentiation of various TSEs. The identification of the E211K PRNP polymorphism in cattle and it’s association with Htype BSE (Greenlee, Smith et al. 2012) may be another another animal model that can contribute to our understanding of hereditary forms of TSEs. Besides the potential to study the onset of spontaneous disease late in life, inoculation of E211K cattle with H-type BSE leads to clinical signs in approximately 10


Figure 4 Biopsy of rectal mucosa in sheep and goats can detect PrPSc accumulation in live animals prior to onset of clinical illness. Mucosa-associated lymphoid follicles are strongly immunoreactive for PrPSc (red reaction product) in a sheep with scrapie. Scale bar = 100 µm.


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thickness (vertical line). Retinal thinning has been associated with accumulation of PrPSc in cattle and sheep with prion diseases. Electroretinography generates a waveform that represents the electrical response of the retina to light (B). Prolonged b-wave implicit time and decreased b-wave amplitude are two parameters that have been associated with retinal accumulation of PrPSc.

months, which greatly reduces the duration normally required to conduct TSE studies in large animals. Prion diseases are related to other neurodegenerative protein misfolding disorders including Parkinson’s disease, Alzheimer’s disease, ALS, and Huntington’s disease (Aguzzi and O’Connor 2010; Prusiner 2012). Common features like self-propagation of misfolded protein and spread throughout neuroanatomic regions of the brain suggest that TSE models (rodent and ruminant) of disease and successful TSE treatment strategies may translate to related neurodegenerative disorders.

Conclusions Our understanding of TSEs has advanced tremendously since prions were first reported to be the disease-causing agent of scrapie in sheep and goats. Misfolding of the prion protein results in similar diseases in cattle, cervids, humans, and other mammalian species. TSEs are transmissible within a species by exposure to or ingestion of infectious material, but the likelihood of transmission between species varies with the TSE. For example, scrapie does not appear to be transmissible to humans, but consumption of BSE contaminated beef can result in developing vCJD. This review highlighted scrapie and BSE, the natural TSEs of small ruminants (sheep and goats) and cattle, respectively. A major difference between scrapie and BSE is host-related differences in distribution of PrPSc accumulation within an animal. Sheep and goats have widespread accumulation of PrPSc throughout the lymphoreticular system, while cattle have very limited accumulation of PrPSc outside of the CNS. This is correlated with the likelihood of transmission within a herd. That is, while transmission of scrapie within a herd of sheep or goats is well described, transmission of BSE from one animal to another in a herd of cattle has not been reported. The difference in distribution of PrPSc accumulation also results in a differential ability to identify infected animals prior to the onset of clinical illness. Rectal mucosal biopsy to detect PrPSc accumulation can be used to identify infected sheep or goats, but is not useful to identify BSE-infected cattle. Finally, scrapie and BSE are vastly different in their risk to human health. While it is well accepted that scrapie is not transmissible to humans, BSE remains a risk to the food supply.

Although much has been learned about scrapie and BSE, there is still a role for livestock in the study of TSEs moving forward. As new strains of these TSEs emerge, work to determine their interspecies transmission will be necessary to constantly reevaluate their risk to the food supply. In addition, TSEs of livestock are a natural, large-animal model of a protein-misfolding disease. Thus, they can provide a valuable step between mouse and human studies on intervention strategies for a whole family of protein misfolding disorders.

Acknowledgments Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity employer.

References Acin C, Martin-Burriel I, Monleon E, Lyahyai J, Pitarch JL, Serrano C, Monzon M, Zaragoza P, Badiola JJ. 2013. Prion protein gene variability in Spanish goats. Inference through susceptibility to classical scrapie strains and pathogenic distribution of peripheral PrPSc. PLoS One 8:e61118. Acutis PL, Bossers A, Priem J, Riina MV, Peletto S, Mazza M, Casalone C, Forloni G, Ru G, Caramelli M. 2006. Identification of prion protein gene polymorphisms in goats from Italian scrapie outbreaks. J Gen Virol 87(Pt 4):1029 – 1033. Aguilar-Calvo P, Espinosa JC, Pintado B, Gutierrez-Adan A, Alamillo E, Miranda A, Prieto I, Bossers A, Andreoletti O, Torres JM. 2014. Role of the goat K222-PrP(C) polymorphic variant in prion infection resistance. J Virol 88:2670–2676. Aguzzi A, O’Connor T. 2010. Protein aggregation diseases: pathogenicity and therapeutic perspectives. Nat Rev Drug Discov 9:237–248. Aguzzi A, Sigurdson C, Heikenwaelder M. 2008. Molecular mechanisms of prion pathogenesis. Annu Rev Pathol 3:11–40. Alverson J, O’Rourke KI, Baszler TV. 2006. PrPSc accumulation in fetal cotyledons of scrapie-resistant lambs is influenced by fetus location in the uterus. J Gen Virol 87(Pt 4):1035–1041.


Figure 5 Assessment of retinal morphology and function. Optical coherence tomography (OCT) generates an image of the retina (A) that can be used to monitor retinal

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cattle and L-type bovine spongiform encephalopathy in a mouse model. Emerg Infect Dis 13:1887–1894. Baron T, Biacabe AG, Arsac JN, Benestad S, Groschup MH. 2007. Atypical transmissible spongiform encephalopathies (TSEs) in ruminants. Vaccine 25:5625–5630. Barria MA, Ironside JW, Head MW. 2014. Exploring the zoonotic potential of animal prion diseases: in vivo and in vitro approaches. Prion 8:85–91. Beck KE, Vickery CM, Lockey R, Holder T, Thorne L, Terry LA, Denyer M, Webb P, Simmons MM, Spiropoulos J. 2012. The interpretation of disease phenotypes to identify TSE strains following murine bioassay: characterisation of classical scrapie. Vet Res 43:77. Bellworthy SJ, Dexter G, Stack M, Chaplin M, Hawkins SA, Simmons MM, Jeffrey M, Martin S, Gonzalez L, Hill P. 2005. Natural transmission of BSE between sheep within an experimental flock. Vet Rec 157:206. Bencsik A, Leboidre M, Debeer S, Aufauvre C, Baron T. 2013. Unique properties of the classical bovine spongiform encephalopathy strain and its emergence from H-type bovine spongiform encephalopathy substantiated by VM transmission studies. J Neuropathol Exp Neurol 72:211–218. Bendheim PE, Brown HR, Rudelli RD, Scala LJ, Goller NL, Wen GY, Kascsak RJ, Cashman NR, Bolton DC. 1992. Nearly ubiquitous tissue distribution of the scrapie agent precursor protein. Neurology 42:149–156. Benestad SL, Arsac JN, Goldmann W, Noremark M. 2008. Atypical/ Nor98 scrapie: properties of the agent, genetics, and epidemiology. Vet Res 39:19. Benestad SL, Sarradin P, Thu B, Schonheit J, Tranulis MA, Bratberg B. 2003. Cases of scrapie with unusual features in Norway and designation of a new type, Nor98. Vet Rec 153: 202–208. Beringue V, Vilotte JL, Laude H. 2008. Prion agent diversity and species barrier. Vet Res 39:47. Bessen RA, Marsh RF. 1992. Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent. J Virol 66:2096–2101. Bessen RA, Marsh RF. 1994. Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy. J Virol 68:7859–7868. Biacabe A-G, Jacobs JG, Bencsik A, Langeveld JPM, Baron TGM. 2007. H-Type bovine spongiform encephalopathy. Prion 1:61–68. Biacabe AG, Laplanche JL, Ryder S, Baron T. 2004. Distinct molecular phenotypes in bovine prion diseases. EMBO Rep 5:110–115. Billinis C, Panagiotidis CH, Psychas V, Argyroudis S, Nicolaou A, Leontides S, Papadopoulos O, Sklaviadis T. 2002. Prion protein gene polymorphisms in natural goat scrapie. J Gen Virol 83(Pt 3):713–721. Brown P, McShane LM, Zanusso G, Detwiler L. 2006. On the question of sporadic or atypical bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. Emerg Infect Dis 12: 1816–1821. Bruce ME, McBride PA, Farquhar CF. 1989. Precise targeting of the pathology of the sialoglycoprotein, PrP, and vacuolar degeneration in mouse scrapie. Neurosci Lett 102:1–6. Bruce ME, Will RG, Ironside JW, McConnell I, Drummond D, Suttie A, McCardle L, Chree A, Hope J, Birkett C, Cousens S, Fraser H, Bostock CJ. 1997. Transmissions to mice indicate that ‘new variant’ CJD is caused by the BSE agent. Nature 389:498–501. Brunelle BW, Hamir AN, Baron T, Biacabe AG, Richt JA, Kunkle RA, Cutlip RC, Miller JM, Nicholson EM. 2007. Polymorphisms of


Andreoletti O, Berthon P, Marc D, Sarradin P, Grosclaude J, van Keulen L, Schelcher F, Elsen JM, Lantier F. 2000. Early accumulation of PrPSc in gut-associated lymphoid and nervous tissues of susceptible sheep from a Romanov flock with natural scrapie. J Gen Virol 81(Pt 12):3115–3126. Andreoletti O, Lacroux C, Chabert A, Monnereau L, Tabouret G, Lantier F, Berthon P, Eychenne F, Lafond-Benestad S, Elsen JM,, Schelcher F. 2002. PrPSc accumulation in placentas of ewes exposed to natural scrapie: influence of foetal PrP genotype and effect on ewe-to-lamb transmission. J Gen Virol 83(Pt 10): 2607–2616. Andreoletti O, Orge L, Benestad SL, Beringue V, Litaise C, Simon S, Le Dur A, Laude H, Simmons H, Lugan S, Corbire F, Costes P, Morel N, Schelcher F, Lacroux C. 2011. Atypical/Nor98 scrapie infectivity in sheep peripheral tissues. PLoS Pathog 7: e1001285. Arnold ME, Ryan JB, Konold T, Simmons MM, Spencer YI, Wear A, Chaplin M, Stack M, Czub S, Mueller R, Webb PR, Davis A, Spiropoulos J, Holdaway J, Hawkins SA, Austin AR, Wells GA. 2007. Estimating the temporal relationship between PrPSc detection and incubation period in experimental bovine spongiform encephalopathy of cattle. J Gen Virol 88(Pt 11):3198–3208. Arnold ME, Wilesmith JW. 2004. Estimation of the age-dependent risk of infection to BSE of dairy cattle in Great Britain. Prev Vet Med 66:35–47. Arsac JN, Andreoletti O, Bilheude JM, Lacroux C, Benestad SL, Baron T. 2007. Similar biochemical signatures and prion protein genotypes in atypical scrapie and Nor98 cases, France and Norway. Emerg Infect Dis 13:58–65. Atarashi R, Wilham JM, Christensen L, Hughson AG, Moore RA, Johnson LM, Onwubiko HA, Priola SA, Caughey B. 2008. Simplified ultrasensitive prion detection by recombinant PrP conversion with shaking. Nat Methods 5:211–212. Baeten LA, Powers BE, Jewell JE, Spraker TR, Miller MW. 2007. A natural case of chronic wasting disease in a free-ranging moose (Alces alces shirasi). J Wildl Dis 43:309–314. Balkema-Buschmann A, Eiden M, Hoffmann C, Kaatz M, Ziegler U, Keller M, Groschup MH. 2011. BSE infectivity in the absence of detectable PrPSc accumulation in the tongue and nasal mucosa of terminally diseased cattle. J Gen Virol 92(Pt 2):467–476. Balkema-Buschmann A, Fast C, Kaatz M, Eiden M, Ziegler U, McIntyre L, Keller M, Hills B, Groschup MH. 2011. Pathogenesis of classical and atypical BSE in cattle. Prev Vet Med 102:112–117. Balkema-Buschmann A, Ziegler U, McIntyre L, Keller M, Hoffmann C, Rogers R, Hills B, Groschup MH. 2011. Experimental challenge of cattle with German atypical bovine spongiform encephalopathy (BSE) isolates. J Toxicol Environ Health A 74:103–109. Bannach O, Reinartz E, Henke F, Dressen F, Oelschlegel A, Kaatz M, Groschup MH, Willbold D, Riesner D, Birkmann E. 2013. Analysis of prion protein aggregates in blood and brain from pre-clinical and clinical BSE cases. Vet Microbiol 166:102–108. Barillet F, Mariat D, Amigues Y, Faugeras R, Caillat H, MoazamiGoudarzi K, Rupp R, Babilliot JM, Lacroux C, Lugan S, Schelcher F, Chartier C, Corbiare F, Androletti O, PerrinChauvineau C. 2009. Identification of seven haplotypes of the caprine PrP gene at codons 127, 142, 154, 211, 222 and 240 in French Alpine and Saanen breeds and their association with classical scrapie. J Gen Virol 90(Pt 3):769–776. Baron T, Bencsik A, Biacabe AG, Morignat E, Bessen RA. 2007. Phenotypic similarity of transmissible mink encephalopathy in


scrapie in five highly infected goat herds. J Gen Virol 94(Pt 1):241–245. Cutlip RC, Miller JM, Hamir AN, Peters J, Robinson MM, Jenny AL, Lehmkuhl HD, Taylor WD, Bisplinghoff FD. 2001. Resistance of cattle to scrapie by the oral route. Can J Vet Res 65:131–132. Cutlip RC, Miller JM, Lehmkuhl HD. 1997. Second passage of a US scrapie agent in cattle. J Comp Pathol 117:271–275. Cutlip RC, Miller JM, Race RE, Jenny AL, Katz JB, Lehmkuhl HD, DeBey BM, Robinson MM. 1994. Intracerebral transmission of scrapie to cattle. J Infect Dis 169:814–820. Dagleish MP, Rodger SM, Simmons MM, Finlayson J, Buxton D, Chianini F. 2008. Atypical scrapie in a sheep in Scotland. Vet Rec 162:518–519. DeArmond SJ, McKinley MP, Barry RA, Braunfeld MB, McColloch JR, Prusiner SB. 1985. Identification of prion amyloid filaments in scrapie-infected brain. Cell 41:221–235. De Bosschere H, Roels S, Dechamps P, Vanopdenbosch E. 2007. TSE detected in a Belgian ARR-homozygous sheep via active surveillance. Vet J 173:449–451. De Bosschere H, Roels S, Vanopdenbosch E. 2004. Atypical case of bovine spongiform encephalopathy in an East-Flemish cow in Belgium. The International Journal of Applied Research 2:52–54. Dennis MM, Thomsen BV, Marshall KL, Hall SM, Wagner BA, Salman MD, Norden DK, Gaiser C, Sutton DL. 2009. Evaluation of immunohistochemical detection of prion protein in rectoanal mucosa-associated lymphoid tissue for diagnosis of scrapie in sheep. Am J Vet Res 70:63–72. Detwiler LA, Baylis M. 2003. The epidemiology of scrapie. Rev Sci Tech 22:121–143. Donnelly CA, Ferguson NM, Ghani AC, Anderson RM. 2002. Implications of BSE infection screening data for the scale of the British BSE epidemic and current European infection levels. Proc Biol Sci 269:2179–2190. Dormont D. 2002. Prion diseases: pathogenesis and public health concerns. FEBS Lett 529:17–21. Dudas S, Yang J, Graham C, Czub M, McAllister TA, Coulthart MB, Czub S. 2010. Molecular, biochemical and genetic characteristics of BSE in Canada. PLoS ONE 5:e10638. Eckroade RJ, ZuRhein GM, Hanson RP. 1973. Transmissible mink encephalopathy in carnivores: clinical, light and electron microscopic studies in raccons, skunks and ferrets. J Wildl Dis 9:229–240. Eckroade RJ, Zu Rhein GM, Marsh RF, Hanson RP. 1970. Transmissible mink encephalopathy: experimental transmission to the squirrel monkey. Science 169:1088–1090. Eloit M, Adjou K, Coulpier M, Fontaine JJ, Hamel R, Lilin T, Messiaen S, Andreoletti O, Baron T, Bencsik A, Biacabe AG, Beringue V, Laude H, Le Dur A, Vilotte JL, Comoy E, Deslys JP, Grassi J, Simon S, Lantier F, Sarradin P. 2005. BSE agent signatures in a goat. Vet Rec 156:523–524. Ersdal C, Ulvund MJ, Espenes A, Benestad SL, Sarradin P, Landsverk T. 2005. Mapping PrPSc propagation in experimental and natural scrapie in sheep with different PrP genotypes. Vet Pathol 42:258–274. Espinosa JC, Morales M, Castilla J, Rogers M, Torres JM. 2007. Progression of prion infectivity in asymptomatic cattle after oral bovine spongiform encephalopathy challenge. J Gen Virol 88 (Pt 4):1379–1383. Fast C, Groschup MH. 2013. Classical and atypical scrapie in sheep and goats. In: Zou W-Q, Gambetti P, eds. Prions and Diseases: Volume 2, Animals, Humans, and the Environment. New York: Springer Science + Business Media.


the prion gene promoter region that influence classical bovine spongiform encephalopathy susceptibility are not applicable to other transmissible spongiform encephalopathies in cattle. J Anim Sci 85:3142–3147. Budka H. 2003. Neuropathology of prion diseases. Br Med Bull 66:121–130. Buschmann A, Biacabe AG, Ziegler U, Bencsik A, Madec JY, Erhardt G, Luhken G, Baron T, Groschup MH. 2004. Atypical scrapie cases in Germany and France are identified by discrepant reaction patterns in BSE rapid tests. J Virol Methods 117:27–36. Buschmann A, Gretzschel A, Biacabe AG, Schiebel K, Corona C, Hoffmann C, Eiden M, Baron T, Casalone C, Groschup MH. 2006. Atypical BSE in Germany-proof of transmissibility and biochemical characterization. Vet Microbiol 117:103–116. Buschmann A, Groschup MH. 2005. Highly bovine spongiform encephalopathy-sensitive transgenic mice confirm the essential restriction of infectivity to the nervous system in clinically diseased cattle. J Infect Dis 192:934–942. Casalone C, Zanusso G, Acutis P, Ferrari S, Capucci L, Tagliavini F, Monaco S, Caramelli M. 2004. Identification of a second bovine amyloidotic spongiform encephalopathy: molecular similarities with sporadic Creutzfeldt-Jakob disease. Proc Natl Acad Sci USA 101:3065–3070. Cassard H, Torres JM, Lacroux C, Douet JY, Benestad SL, Lantier F, Lugan S, Lantier I, Costes P, Aron N, Reine F, Herzog L, Espinosa JC, Beringue V, Andréoletti O. 2014. Evidence for zoonotic potential of ovine scrapie prions. Nat Commun 5:5821. Castilla J, Gutierrez Adan A, Brun A, Pintado B, Ramirez MA, Parra B, Doyle D, Rogers M, Salguero FJ, Sanchez C, SanchezVizcaíno JM, Torres JM. 2003. Early detection of PrPres in BSEinfected bovine PrP transgenic mice. Arch Virol 148:677–691. Caughey B, Neary K, Buller R, Ernst D, Perry LL, Chesebro B, Race RE. 1990. Normal and scrapie-associated forms of prion protein differ in their sensitivities to phospholipase and proteases in intact neuroblastoma cells. J Virol 64:1093–1101. Caughey B, Raymond GJ. 1991. The scrapie-associated form of PrP is made from a cell surface precursor that is both proteaseand phospholipase-sensitive. J Biol Chem 266:18217–18223. Clark WW, Hourrigan JL, Hadlow WJ. 1995. Encephalopathy in cattle experimentally infected with the scrapie agent. Am J Vet Res 56:606–612. Clawson ML, Heaton MP, Keele JW, Smith TP, Harhay GP, Laegreid WW. 2006. Prion gene haplotypes of U.S. cattle. BMC Genet 7:51. Cobb NJ, Surewicz WK. 2009. Prion diseases and their biochemical mechanisms. Biochemistry 48:2574–2585. Collinge J, Clarke AR. 2007. A general model of prion strains and their pathogenicity. Science 318:930–936. Collinge J, Palmer MS, Dryden AJ. 1991. Genetic predisposition to iatrogenic Creutzfeldt-Jakob disease. Lancet 337:1441–1442. Collinge J, Sidle KC, Meads J, Ironside J, Hill AF. 1996. Molecular analysis of prion strain variation and the aetiology of ‘new variant’ CJD. Nature 383:685–690. Colussi S, Vaccari G, Maurella C, Bona C, Lorenzetti R, Troiano P, Casalinuovo F, Di Sarno A, Maniaci MG, Zuccon F, Nonno R, Casalone C, Mazza M, Ru G, Caramelli M, Agrimi U, Acutis PL. 2008. Histidine at codon 154 of the prion protein gene is a risk factor for Nor98 scrapie in goats. J Gen Virol 89(Pt 12): 3173–3176. Corbiere F, Perrin-Chauvineau C, Lacroux C, Costes P, Thomas M, Bremaud I, Martin S, Lugan S, Chartier C, Schelcher F, Barillet F, Andreoletti O. 2013. PrP-associated resistance to

| 19

20 |


caprine PrP gene: a codon 142 mutation associated with scrapie incubation period. J Gen Virol 77(Pt 11):2885–2891. Goldmann W, Ryan K, Stewart P, Parnham D, Xicohtencatl R, Fernandez N, Saunders G, Windl O, Gonzalez L, Bossers A, Foster J. 2011. Caprine prion gene polymorphisms are associated with decreased incidence of classical scrapie in goat herds in the United Kingdom. Vet Res 42:110. Gombojav A, Shimauchi I, Horiuchi M, Ishiguro N, Shinagawa M, Kitamoto T, Miyoshi I, Mohri S, Takata M. 2003. Susceptibility of transgenic mice expressing chimeric sheep, bovine and human PrP genes to sheep scrapie. J Vet Med Sci 65:341–347. Gonzalez L, Dagleish MP, Martin S, Dexter G, Steele P, Finlayson J, Jeffrey M. 2008. Diagnosis of preclinical scrapie in live sheep by the immunohistochemical examination of rectal biopsies. Vet Rec 162:397–403. Gonzalez L, Horton R, Ramsay D, Toomik R, Leathers V, Tonelli Q, Dagleish MP, Jeffrey M, Terry L. 2008. Adaptation and evaluation of a rapid test for the diagnosis of sheep scrapie in samples of rectal mucosa. J Vet Diagn Invest 20:203–208. Gonzalez L, Jeffrey M, Dagleish MP, Goldmann W, Siso S, Eaton SL, Martin S, Finlayson J, Stewart P, Steele P, Pang Y, Hamilton S, Reid HW, Chianini F. 2012. Susceptibility to scrapie and disease phenotype in sheep: cross-PRNP genotype experimental transmissions with natural sources. Vet Res 43:55. Gonzalez L, Martin S, Begara-McGorum I, Hunter N, Houston F, Simmons M, Jeffrey M. 2002. Effects of agent strain and host genotype on PrP accumulation in the brain of sheep naturally and experimentally affected with scrapie. J Comp Pathol 126:17–29. Gonzalez L, Martin S, Hawkins SA, Goldmann W, Jeffrey M, Siso S. 2010. Pathogenesis of natural goat scrapie: modulation by host PRNP genotype and effect of co-existent conditions. Vet Res 41:48. Gonzalez L, Martin S, Siso S, Konold T, Ortiz-Pelaez A, Phelan L, Goldmann W, Stewart P, Saunders G, Windl O, Jeffrey M, Hawkins SA, Dawson M, Hope J. 2009. High prevalence of scrapie in a dairy goat herd: tissue distribution of diseaseassociated PrP and effect of PRNP genotype and age. Vet Res 40:65. Gordon WS. 1946. Advances in veterinary research: Louping-ill, tick-boune fever and scrapie. Vet Rec 58:516–525. Gough KC, Maddison BC. 2010. Prion transmission: prion excretion and occurrence in the environment. Prion 4:275–282. Gray JG, Dudas S, Graham C, Czub S. 2012. Performance analysis of rapid diagnostic tests on atypical bovine spongiform encephalopathy. J Vet Diagn Invest 24:976–980. Greenlee JJ, Hamir AN, West Greenlee MH. 2006. Abnormal prion accumulation associated with retinal pathology in experimentally inoculated scrapie-affected sheep. Vet Pathol 43:733–739. Greenlee JJ, Nicholson EM, Smith JD, Kunkle RA, Hamir AN. 2012. Susceptibility of cattle to the agent of chronic wasting disease from elk after intracranial inoculation. J Vet Diagn Invest 24:1087–1093. Greenlee JJ, Smith JD, Kunkle RA. 2011. White-tailed deer are susceptible to the agent of sheep scrapie by intracerebral inoculation. Vet Res 42:107. Greenlee JJ, Smith JD, West Greenlee MH, Nicholson EM. 2012. Clinical and pathologic features of H-type bovine spongiform encephalopathy associated with E211K prion protein polymorphism. PLoS ONE 7:e38678. Greenlee JJ, Zhang X, Nicholson EM, Kunkle RA, Hamir AN. 2012c. Prolonged incubation time in sheep with prion protein containing lysine at position 171. J Vet Diagn Invest 24:554–558.


Fast C, Keller M, Balkema-Buschmann A, Hills B, Groschup MH. 2013. Complementary studies detecting classical bovine spongiform encephalopathy infectivity in jejunum, ileum and ileocaecal junction in incubating cattle. Vet Res 44:123. Fediaevsky A, Maurella C, Noremark M, Ingravalle F, Thorgeirsdottir S, Orge L, Poizat R, Hautaniemi M, Liam B, Calavas D. 2010. The prevalence of atypical scrapie in sheep from positive flocks is not higher than in the general sheep population in 11 European countries. BMC Vet Res 6:9. Fediaevsky A, Tongue SC, Noremark M, Calavas D, Ru G, Hopp P. 2008. A descriptive study of the prevalence of atypical and classical scrapie in sheep in 20 European countries. BMC Vet Res 4:19. Fernandez-Borges N, Erana H, Elezgarai SR, Harrathi C, Gayosso M, Castilla J. 2013. Infectivity versus seeding in neurodegenerative diseases sharing a prion-like mechanism. Int J Cell Biol 2013:583498. Foster JD, Goldmann W, McKenzie C, Smith A, Parnham DW, Hunter N. 2004. Maternal transmission studies of BSE in sheep. J Gen Virol 85(Pt 10):3159–3163. Foster JD, Hope J, Fraser H. 1993. Transmission of bovine spongiform encephalopathy to sheep and goats. Vet Rec 133:339–341. Foster JD, Parnham D, Chong A, Goldmann W, Hunter N. 2001. Clinical signs, histopathology and genetics of experimental transmission of BSE and natural scrapie to sheep and goats. Vet Rec 148:165–171. Foster JD, Parnham DW, Hunter N, Bruce M. 2001. Distribution of the prion protein in sheep terminally affected with BSE following experimental oral transmission. J Gen Virol 82(Pt 10):2319–2326. Franz M, Eiden M, Balkema-Buschmann A, Greenlee J, Schatzl H, Fast C, Richt J, Hildebrandt JP, Groschup MH. 2012. Detection of PrPSc in peripheral tissues of clinically affected cattle after oral challenge with bovine spongiform encephalopathy. J Gen Virol 93(Pt 12):2740–2748. Fraser H, Dickinson AG. 1968. The sequential development of the brain lesion of scrapie in three strains of mice. J Comp Pathol 78:301–311. Fraser H, Dickinson AG. 1973. Scrapie in mice. Agent-strain differences in the distribution and intensity of grey matter vacuolation. J Comp Pathol 83:29–40. Fréchette J-D. 2005. Mad Cow Disease in Canada: An economic overview. [Internet]. Parliament of Canada; [cited March 13, 2015]. Available from: http://www.parl.gc.ca/Content/LOP/ ResearchPublications/tips/tip116-e.htm. Gavier-Widen D, Noremark M, Benestad S, Simmons M, Renstrom L, Bratberg B, Elvander M, af Segerstad CH. 2004. Recognition of the Nor98 variant of ccrapie in the Swedish sheep population. J Vet Diagn Invest 16:562–567 Gill ON, Spencer Y, Richard-Loendt A, Kelly C, Dabaghian R, Boyes L, Linehan J, Simmons M, Webb P, Bellerby P, Andrews N, Hilton DA, Ironside JW, Beck J, Poulter M, Mead S, Brandner S. 2013. Prevalent abnormal prion protein in human appendixes after bovine spongiform encephalopathy epizootic: large scale survey. BMJ 347:f5675. Goldmann W, Hunter N, Foster JD, Salbaum JM, Beyreuther K, Hope J. 1990. Two alleles of a neural protein gene linked to scrapie in sheep. Proc Natl Acad Sci USA 87:2476–2480. Goldmann W, Hunter N, Smith G, Foster J, Hope J. 1994. 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(Pt 5):989–995. Goldmann W, Martin T, Foster J, Hughes S, Smith G, Hughes K, Dawson M, Hunter N. 1996. Novel polymorphisms in the


visual pathways associated to natural Scrapie in sheep. Brain Res 1108:188–194. Hunter N, Foster JD, Benson G, Hope J. 1991. Restriction fragment length polymorphisms of the scrapie-associated fibril protein (PrP) gene and their association with susceptibility to natural scrapie in British sheep. J Gen Virol 72(Pt 6):1287–1292. Hunter N, Houston F, Foster J, Goldmann W, Drummond D, Parnham D, Kennedy I, Green A, Stewart P, Chong A. 2012. Susceptibility of young sheep to oral infection with bovine spongiform encephalopathy decreases significantly after weaning. J Virol 86:11856–11862. Jeffrey M, Gonzalez L. 2004. Pathology and pathogenesis of bovine spongiform encephalopathy and scrapie. Curr Top Microbiol Immunol 284:65–97. Jeffrey M, Gonzalez L, Espenes A, Press C, Martin S, Chaplin M, Davis L, Landsverk T, Macaldowie C, Eaton S, McGovern G. 2006. Transportation of prion protein across the intestinal mucosa of scrapie-susceptible and scrapie-resistant sheep. J Pathol 209:4–14. Jeffrey M, Martin S, Gonzalez L, Ryder SJ, Bellworthy SJ, Jackman R. 2001. Differential diagnosis of infections with the bovine spongiform encephalopathy (BSE) and scrapie agents in sheep. J Comp Pathol 125:271–284. Jeffrey M, McGovern G, Goodsir CM, Brown KL, Bruce ME. 2000. Sites of prion protein accumulation in scrapie-infected mouse spleen revealed by immuno-electron microscopy. J Pathol 191:323–332. Jeffrey M, Ryder S, Martin S, Hawkins SA, Terry L, BerthelinBaker C, Bellworthy SJ. 2001. Oral inoculation of sheep with the agent of bovine spongiform encephalopathy (BSE). 1. Onset and distribution of disease-specific PrP accumulation in brain and viscera. J Comp Pathol 124:280–289. Jones M, Peden AH, Prowse CV, Groner A, Manson JC, Turner ML, Ironside JW, MacGregor IR, Head MW. 2007. In vitro amplification and detection of variant Creutzfeldt-Jakob disease PrPSc. J Pathol 213:21–26. Kimberlin RH. 1992. Bovine spongiform encephalopathy. Rev Sci Tech 11:347–489. Kitamoto T, Nakamura K, Nakao K, Shibuya S, Shin RW, Gondo Y, Katsuki M, Tateishi J. 1996. Humanized prion protein knock-in by Cre-induced site-specific recombination in the mouse. Biochem Biophys Res Commun 222:742–747. Kittelberger R, Chaplin MJ, Simmons MM, Ramirez-Villaescusa A, McIntyre L, MacDiarmid SC, Hannah MJ, Jenner J, Bueno R, Bayliss D, Black H, Pigott CJ, O’Keefe JS. 2010. Atypical scrapie/Nor98 in a sheep from New Zealand. J Vet Diagn Invest 22:863–875. Klingeborn M, Wik L, Simonsson M, Renstrom LH, Ottinger T, Linne T. 2006. Characterization of proteinase K-resistant Nand C-terminally truncated PrP in Nor98 atypical scrapie. J Gen Virol 87(Pt 6):1751–1760. Kong Q, Zheng M, Casalone C, Qing L, Huang S, Chakraborty B, Wang P, Chen F, Cali I, Corona C, Martucci F, Iulini B, Acutis P, Wang L, Liang J, Wang M, Li X, Monaco S, Zanusso G, Zou WQ, Caramelli M, Gambetti P. 2008. Evaluation of the human transmission risk of an atypical bovine spongiform encephalopathy prion strain. J Virol 82:3697–3701. Konold T, Bone GE, Clifford D, Chaplin MJ, Cawthraw S, Stack MJ, Simmons MM. 2012. Experimental H-type and L-type bovine spongiform encephalopathy in cattle: observation of two clinical syndromes and diagnostic challenges. BMC Vet Res 8:22. Konold T, Lee YH, Stack MJ, Horrocks C, Green RB, Chaplin M, Simmons MM, Hawkins SA, Lockey R, Spiropoulos J, Wilesmith JW, Wells GA. 2006. Different prion disease phenotypes


Griffiths PC, Spiropoulos J, Lockey R, Tout AC, Jayasena D, Plater JM, Chave A, Green RB, Simonini S, Thorne L, Dexter I, Balkema-Buschmann A, Groschup MH, Baringue V, Le Dur A, Laude H, Hope J. 2010. Characterization of atypical scrapie cases from Great Britain in transgenic ovine PrP mice. J Gen Virol 91(Pt 8):2132–2138. Hadlow WJ, Race RE, Kennedy RC. 1987. Experimental infection of sheep and goats with transmissible mink encephalopathy virus. Can J Vet Res 51:135–144. Hamir AN, Cutlip RC, Miller JM, Williams ES, Stack MJ, Miller MW, O’Rourke KI, Chaplin MJ. 2001. Preliminary findings on the experimental transmission of chronic wasting disease agent of mule deer to cattle. J Vet Diagn Invest 13:91–96. Hamir AN, Kehrli ME, Kunkle RA, Greenlee JJ, Nicholson EM, Richt JA, Miller JM, Cutlip RC. 2011. Experimental interspecies transmission studies of the transmissible spongiform encephalopathies to cattle: comparison to bovine spongiform encephalopathy in cattle. J Vet Diagn Invest 23:407–420. Hamir AN, Kunkle RA, Cutlip RC, Miller JM, O’Rourke KI, Williams ES, Miller MW, Stack MJ, Chaplin MJ, Richt JA. 2005. Experimental transmission of chronic wasting disease agent from mule deer to cattle by the intracerebral route. J Vet Diagn Invest 17:276–281. Hamir AN, Kunkle RA, Cutlip RC, Miller JM, Williams ES, Richt JA. 2006. Transmission of chronic wasting disease of mule deer to Suffolk sheep following intracerebral inoculation. J Vet Diagn Invest 18:558–565. Hamir AN, Kunkle RA, Miller JM, Bartz JC, Richt JA. 2006. First and second cattle passage of transmissible mink encephalopathy by intracerebral inoculation. Vet Pathol 43:118–126. Hamir AN, Kunkle RA, Miller JM, Greenlee JJ, Richt JA. 2006. Experimental second passage of chronic wasting disease (CWDmule deer ) agent to cattle. J Comp Pathol 134:63–69. Hamir AN, Miller JM, Kunkle RA, Hall SM, Richt JA. 2007. Susceptibility of cattle to first-passage intracerebral inoculation with chronic wasting disease agent from white-tailed deer. Vet Pathol 44:487–493. Hayashi HK, Yokoyama T, Takata M, Iwamaru Y, Imamura M, Ushiki YK, Shinagawa M. 2005. The N-terminal cleavage site of PrPSc from BSE differs from that of PrPSc from scrapie. Biochem Biophys Res Commun 328:1024–1027. Heggebo R, Press CM, Gunnes G, Lie KI, Tranulis MA, Ulvund M, Groschup MH, Landsverk T. 2000. 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(Pt 9):2327–2337. Hetz C, Russelakis-Carneiro M, Maundrell K, Castilla J, Soto C. 2003. Caspase-12 and endoplasmic reticulum stress mediate neurotoxicity of pathological prion protein. Embo J 22:5435–5445. Hill AF, Collinge J. 2001. Strain variations and species barriers. Contrib Microbiol 7:48–57. Hill AF, Collinge J. 2004. Prion strains and species barriers. Contrib Microbiol 11:33–49. Hill AF, Desbruslais M, Joiner S, Sidle KC, Gowland I, Collinge J, Doey LJ, Lantos P. 1997. The same prion strain causes vCJD and BSE. Nature 389:448–50, 526. Hope J, Wood SC, Birkett CR, Chong A, Bruce ME, Cairns D, Goldmann W, Hunter N, Bostock CJ. 1999. Molecular analysis of ovine prion protein identifies similarities between BSE and an experimental isolate of natural scrapie, CH1641. J Gen Virol 80(Pt 1):1–4. Hortells P, Monzon M, Monleon E, Acin C, Vargas A, Bolea R, Lujan L, Badiola JJ. 2006. Pathological findings in retina and

| 21

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transmissible mink encephalopathy agent. J Infect Dis 120:713–719. Marsh RF, Hartsough G. R. 1988. Evidence that Transmissible Mink Encephalopathy Results from feeding infected cattle. Proceedings of the 4th International Scientific Congress in Fur Animal Production; August 21–28, 1988. Toronto, Canada. McKinley MP, Bolton DC, Prusiner SB. 1983. A protease-resistant protein is a structural component of the scrapie prion. Cell 35:57–62. Mitchell GB, O’Rourke KI, Harrington NP, Soutyrine A, Simmons MM, Dudas S, Zhuang D, Laude H, Balachandran A. 2010. Identification of atypical scrapie in Canadian sheep. J Vet Diagn Invest 22:408–411. Moda F, Gambetti P, Notari S, Concha-Marambio L, Catania M, Park KW, Maderna E, Suardi S, Haik S, Brandel JP, Ironside J, Knight R, Tagliavini F, Soto C. 2014. Prions in the urine of patients with variant Creutzfeldt-Jakob disease. N Engl J Med 371:530–539. Monleon E, Garza MC, Sarasa R, Alvarez-Rodriguez J, Bolea R, Monzon M, Vargas MA, Badiola JJ, Acin C. 2011. An assessment of the efficiency of PrPSc detection in rectal mucosa and thirdeyelid biopsies from animals infected with scrapie. Vet Microbiol 147:237–243. Moore RA, Vorberg I, Priola SA. 2005. Species barriers in prion diseases–brief review. Arch Virol Suppl 19:187–202. Morales R, Abid K, Soto C. 2007. The prion strain phenomenon: molecular basis and unprecedented features. Biochim Biophys Acta 1772:681–691. Muramatsu Y, Onodera A, Horiuchi M, Ishiguro N, Shinagawa M. 1994. Detection of PrPSc in sheep at the preclinical stage of scrapie and its significance for diagnosis of insidious infection. Arch Virol 134:427–432. Murayama Y, Yoshioka M, Masujin K, Okada H, Iwamaru Y, Imamura M, Matsuura Y, Fukuda S, Onoe S, Yokoyama T. 2010. Sulfated dextrans enhance in vitro amplification of bovine spongiform encephalopathy PrP(Sc) and enable ultrasensitive detection of bovine PrP(Sc). PLoS One 5:e13152. NCBA. 2014. BSEinfo.org [Internet]. Available online (http://www. bseinfo.org/bsediagnosis.aspx), accessed on March 10, 2015. Nicholson EM, Brunelle BW, Richt JA, Kehrli ME Jr., Greenlee JJ. 2008. Identification of a heritable polymorphism in bovine PRNP associated with genetic transmissible spongiform encephalopathy: evidence of heritable BSE. PLoS ONE 3:e2912. Oesch B, Westaway D, Walchli M, McKinley MP, Kent SB, Aebersold R, Barry RA, Tempst P, Teplow DB, Hood LE. 1985. A cellular gene encodes scrapie PrP 27–30 protein. Cell 40:735–746. Okada H, Iwamaru Y, Imamura M, Masujin K, Matsuura Y, Shimizu Y, Kasai K, Mohri S, Yokoyama T, Czub S. 2011. Experimental H-type bovine spongiform encephalopathy characterized by plaques and glial- and stellate-type prion protein deposits. Vet Res 42:79. Okada H, Murayama Y, Shimozaki N, Yoshioka M, Masujin K, Imamura M, Iwamaru Y, Matsuura Y, Miyazawa K, Fukuda S, Yokoyama T, Mohri S. 2012. Prion in saliva of bovine spongiform encephalopathy-infected cattle. Emerg Infect Dis 18:2091–2092. O’Rourke KI, Baszler TV, Besser TE, Miller JM, Cutlip RC, Wells GA, Ryder SJ, Parish SM, Hamir AN, Cockett NE. 2000. Preclinical diagnosis of scrapie by immunohistochemistry of third eyelid lymphoid tissue. J Clin Microbiol 38:3254–3259. O’Rourke KI, Holyoak GR, Clark WW, Mickelson JR, Wang S, Melco RP, Besser TE, Foote WC. 1997. PrP genotypes and experimental scrapie in orally inoculated Suffolk sheep in the United States. J Gen Virol 78(Pt 4):975–978.


result from inoculation of cattle with two temporally separated sources of sheep scrapie from Great Britain. BMC Vet Res 2:31. Konold T, Moore SJ, Bellworthy SJ, Simmons HA. 2008. Evidence of scrapie transmission via milk. BMC Vet Res 4:14. Konold T, Moore SJ, Bellworthy SJ, Terry LA, Thorne L, Ramsay A, Salguero FJ, Simmons MM, Simmons HA. 2013. Evidence of effective scrapie transmission via colostrum and milk in sheep. BMC Vet Res 9:99. Kovacs GG, Puopolo M, Ladogana A, Pocchiari M, Budka H, van Duijn C, Collins SJ, Boyd A, Giulivi A, Coulthart M, DelasnerieLaupretre N, Brandel JP, Zerr I, Kretzschmar HA, de PedroCuesta J, Calero-Lara M, Glatzel M, Aguzzi A, Bishop M, Knight R, Belay G, Will R, Mitrova E. 2005. Genetic prion disease: the EUROCJD experience. Hum Genet 118:166–174. Kupfer L, Eiden M, Buschmann A, Groschup MH. 2007. Amino acid sequence and prion strain specific effects on the in vitro and in vivo convertibility of ovine/murine and bovine/murine prion protein chimeras. Biochim Biophys Acta 1772:704–713. Lacroux C, Comoy E, Moudjou M, Perret-Liaudet A, Lugan S, Litaise C, Simmons H, Jas-Duval C, Lantier I, Beringue V. 2014. Preclinical detection of variant CJD and BSE prions in blood. PLoS Pathog 10:e1004202. Lacroux C, Perrin-Chauvineau C, Corbiere F, Aron N, AguilarCalvo P, Torres JM, Costes P, Bremaud I, Lugan S, Schelcher F. 2014. Genetic resistance to scrapie infection in experimentally challenged goats. J Virol 88:2406–2413. Laegreid WW, Clawson ML, Heaton MP, Green BT, O’Rourke KI, Knowles DP. 2008. Scrapie resistance in ARQ sheep. J Virol 82:10318–10320. Langeveld JP, Erkens JH, Rammel I, Jacobs JG, Davidse A, van Zijderveld FG, Bossers A, Schildorfer H. 2011. Four independent molecular prion protein parameters for discriminating new cases of C, L, and H bovine spongiform encephalopathy in cattle. J Clin Microbiol 49:3026–3028. Langeveld JP, Jacobs JG, Erkens JH, Bossers A, van Zijderveld FG, van Keulen LJ. 2006. Rapid and discriminatory diagnosis of scrapie and BSE in retro-pharyngeal lymph nodes of sheep. BMC Vet Res 2:19. Laplanche JL, Chatelain J, Westaway D, Thomas S, Dussaucy M, Brugere-Picoux J, Launay JM. 1993. PrP polymorphisms associated with natural scrapie discovered by denaturing gradient gel electrophoresis. Genomics 15:30–37. Le Dur A, Beringue V, Andreoletti O, Reine F, Lai TL, Baron T, Bratberg B, Vilotte JL, Sarradin P, Benestad SL, Laude H. 2005. A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes. Proc Natl Acad Sci USA 102:16031–6. Legname G, Nguyen HO, Baskakov IV, Cohen FE, Dearmond SJ, Prusiner SB. 2005. Strain-specified characteristics of mouse synthetic prions. Proc Natl Acad Sci USA 102:2168–2173. Levavasseur E, Privat N, Martin JC, Simoneau S, Baron T, Flan B, Torres JM, Haik S. 2014. Molecular modeling of prion transmission to humans. Viruses 6:3766–3777. Loiacono CM, Thomsen BV, Hall SM, Kiupel M, Sutton D, O’Rourke K, Barr B, Anthenill L, Keane D. 2009. Nor98 scrapie identified in the United States. J Vet Diagn Invest 21:454–463. Mabbott NA, Bruce ME. 2002. Follicular dendritic cells as targets for intervention in transmissible spongiform encephalopathies. Semin Immunol 14:285–293. Marsh RF, Bessen RA, Lehmann S, Hartsough GR. 1991. Epidemiological and experimental studies on a new incident of transmissible mink encephalopathy. J Gen Virol 72:589–594. Marsh RF, Burger D, Eckroade R, Zu Rhein GM, Hanson RP. 1969. A preliminary report on the experimental host range of the


Ryder S, Dexter G, Bellworthy S, Tongue S. 2004. Demonstration of lateral transmission of scrapie between sheep kept under natural conditions using lymphoid tissue biopsy. Res Vet Sci 76:211–217. Saa P, Castilla J, Soto C. 2006. Ultra-efficient replication of infectious prions by automated protein misfolding cyclic amplification. J Biol Chem 281:35245–35252. Saba R, Booth SA. 2013. The genetics of susceptibility to variant Creutzfeldt-Jakob disease. Public Health Genom 16:17–24. Saegerman C, Speybroeck N, Roels S, Vanopdenbosch E, Thiry E, Berkvens D. 2004. Decision support tools for clinical diagnosis of disease in cows with suspected bovine spongiform encephalopathy. J Clin Microbiol 42:172–178. Safar J, Wille H, Itri V, Groth D, Serban H, Torchia M, Cohen FE, Prusiner SB. 1998. Eight prion strains have PrP(Sc) molecules with different conformations. Nat Med 4:1157–1165. Sarradin P, Melo S, Barc C, Lecomte C, Andreoletti O, Lantier F, Dacheux JL, Gatti JL. 2008. Semen from scrapie-infected rams does not transmit prion infection to transgenic mice. Reproduction 135:415–418. Saunders GC, Cawthraw S, Mountjoy SJ, Hope J, Windl O. 2006. PrP genotypes of atypical scrapie cases in Great Britain. J Gen Virol 87(Pt 11):3141–3149. Saunders GC, Lantier I, Cawthraw S, Berthon P, Moore SJ, Arnold ME, Windl O, Simmons MM, Andreoletti O, Bellworthy S, Lantier F. 2009. Protective effect of the T112 PrP variant in sheep challenged with bovine spongiform encephalopathy. J Gen Virol 90 (Pt 10):2569–2574. Schelzke G, Kretzschmar HA, Zerr I. 2012. Clinical aspects of common genetic Creutzfeldt-Jakob disease. Eur J Epidemiol 27: 147–149. Schneider K, Fangerau H, Michaelsen B, Raab WH. 2008. The early history of the transmissible spongiform encephalopathies exemplified by scrapie. Brain Res Bull 77:343–355. Schreuder BE, van Keulen LJ, Vromans ME, Langeveld JP, Smits MA. 1998. Tonsillar biopsy and PrPSc detection in the preclinical diagnosis of scrapie. Vet Rec 142:564–568. Scott M, Groth D, Foster D, Torchia M, Yang SL, DeArmond SJ, Prusiner SB. 1993. Propagation of prions with artificial properties in transgenic mice expressing chimeric PrP genes. Cell 73:979–988. Scott AC, Wells GA, Stack MJ, White H, Dawson M. 1990. Bovine spongiform encephalopathy: detection and quantitation of fibrils, fibril protein (PrP) and vacuolation in brain. Vet Microbiol 23:295–304. Seuberlich T. 2007. Atypical scrapie in a Swiss goat and implications for transmissible spongiform encephalopathy surveillance. J Vet Diagn Invest 19:2–8. Sigurdson CJ, Nilsson KP, Hornemann S, Manco G, Polymenidou M, Schwarz P, Leclerc M, Hammarstrom P, Wuthrich K, Aguzzi A. 2007. Prion strain discrimination using luminescent conjugated polymers. Nat Methods 4:1023–1030. Sikorska B, Liberski PP. 2012. Human prion diseases: from Kuru to variant Creutzfeldt-Jakob disease. Subcell Biochem 65:457–496. Simmons MM, Konold T, Simmons HA, Spencer YI, Lockey R, Spiropoulos J, Everitt S, Clifford D. 2007. Experimental transmission of atypical scrapie to sheep. BMC Vet Res 3:20. Simmons MM, Moore SJ, Konold T, Thurston L, Terry LA, Thorne L, Lockey R, Vickery C, Hawkins SA, Chaplin MJ, Spiropoulos J. 2011. Experimental oral transmission of atypical scrapie to sheep. Emerg Infect Dis 17:848–854. Smith JD, Greenlee JJ. 2014. Detection of misfolded prion protein in retina samples of sheep and cattle by use of a commercially available enzyme immunoassay. Am J Vet Res 75:268–272.


O’Rourke KI, Zhuang D, Truscott TC, Yan H, Schneider DA. 2011. Sparse PrPSc accumulation in the placentas of goats with naturally acquired scrapie. BMC Vet Res 7:7. Orru CD, Wilham JM, Vascellari S, Hughson AG, Caughey B. 2012. New generation QuIC assays for prion seeding activity. Prion 6:147–152. Pan KM, Baldwin M, Nguyen J, Gasset M, Serban A, Groth D, Mehlhorn I, Huang Z, Fletterick RJ, Cohen FE. 1993. Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci USA 90:10962–6. Papasavva-Stylianou P, Kleanthous M, Toumazos P, Mavrikiou P, Loucaides P. 2007. Novel polymorphisms at codons 146 and 151 in the prion protein gene of Cyprus goats, and their association with natural scrapie. Vet J 173:459–462. Papasavva-Stylianou P, Windl O, Saunders G, Mavrikiou P, Toumazos P, Kakoyiannis C. 2011. PrP gene polymorphisms in Cyprus goats and their association with resistance or susceptibility to natural scrapie. Vet J 187:245–250. Pattison IH, Millson GC. 1961. Scrapie produced experimentally in goats with special reference to the clinical syndrome. J Comp Pathol 71:101–109. Peretz D, Williamson RA, Legname G, Matsunaga Y, Vergara J, Burton DR, DeArmond SJ, Prusiner SB, Scott MR. 2002. A change in the conformation of prions accompanies the emergence of a new prion strain. Neuron 34:921–932. Porter DD, Porter HG, Cox NA. 1973. Failure to demonstrate a humoral immune response to scrapie infection in mice. J Immunol 111:1407–1410. Press CM, Heggebo R, Espenes A. 2004. Involvement of gut-associated lymphoid tissue of ruminants in the spread of transmissible spongiform encephalopathies. Adv Drug Deliv Rev 56:885–899. Prinz M, Huber G, Macpherson AJ, Heppner FL, Glatzel M, Eugster HP, Wagner N, Aguzzi A. 2003. Oral prion infection requires normal numbers of Peyer’s patches but not of enteric lymphocytes. Am J Pathol 162:1103–1111. Prusiner SB. 1991. Molecular biology of prion diseases. Science 252:1515–1522. Prusiner SB. 2001. Shattuck lecture–neurodegenerative diseases and prions. N Engl J Med 344:1516–1526. Prusiner SB. 2012. Cell biology. A unifying role for prions in neurodegenerative diseases. Science 336:1511–1513. Prusiner SB. 2013. Biology and genetics of prions causing neurodegeneration. Annu Rev Genet 47:601–623. Prusiner SB, Scott MR, DeArmond SJ, Cohen FE. 1998. Prion protein biology. Cell 93:337–348. Race R, Ernst D, Jenny A, Taylor W, Sutton D, Caughey B. 1992. Diagnostic implications of detection of proteinase K-resistant protein in spleen, lymph nodes, and brain of sheep. Am J Vet Res 53:883–889. Richt JA, Hall SM. 2008. BSE case associated with prion protein gene mutation. PLoS Pathog 4:e1000156. Richt JA, Kunkle RA, Alt D, Nicholson EM, Hamir AN, Czub S, Kluge J, Davis AJ, Hall SM. 2007. Identification and characterization of two bovine spongiform encephalopathy cases diagnosed in the United States. J Vet Diagn Invest 19:142–154. Robinson MM, Hadlow WJ, Knowles DP, Huff TP, Lacy PA, Marsh RF, Gorham JR. 1995. Experimental infection of cattle with the agents of transmissible mink encephalopathy and scrapie. J Comp Pathol 113:241–251. Rubenstein R, Bulgin MS, Chang B, Sorensen-Melson S, Petersen RB, LaFauci G. 2012. PrP(Sc) detection and infectivity in semen from scrapie-infected sheep. J Gen Virol 93(Pt 6):1375–1383.

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PMSEqNgNaSJgEygEiAu_XiCuTFRmN5Nz7-QgijJEBXvwio28 Fayed6rkQeLjrQmYeKnuADmc3ViL1F3iyxNwnQDLM3xZj QBA1jAQ2_RtVY8BiLIuD1_GgH_5YMUD3MdWXCHasfG4aJsUTawnAnesDq_8aFgQ5HP975ZvNEF0Y9azzGnWjc6qm GAIdy_gV_eC_BDrGtO2TMqU1JJ0gup0N6R/?1dmy&urile= wcm%3apath%3a%2Faphis_content_library%2Fsa_our_focus %2Fsa_animal_health%2Fsa_animal_disease_information% 2Fsa_sheep_goat_health%2Fsa_scrapie%2Fct_scrapie_home), accessed on March 10, 2015. U.S. Department of Agriculture Food Safety and Inspection Service. 2014. Bovine Spongiform Encephalopathy (BSE) and Specified Risk Material (SRM) Guidance Materials and Resources [Internet]. Available online (http://www.fsis.usda. gov/wps/portal/fsis/topics/regulatory-compliance/specifiedrisk-material), accessed on March 15, 2015. Vaccari G, Di Bari MA, Morelli L, Nonno R, Chiappini B, Antonucci G, Marcon S, Esposito E, Fazzi P, Palazzini N, Troiano P, Petrella A, Di Guardo G, Agrimi U. 2006. Identification of an allelic variant of the goat PrP gene associated with resistance to scrapie. J Gen Virol 87(Pt 5):1395–1402. Vanik DL, Surewicz KA, Surewicz WK. 2004. Molecular basis of barriers for interspecies transmissibility of mammalian prions. Mol Cell 14:139–145. van Keulen LJ, Vromans ME, van Zijderveld FG. 2002. Early and late pathogenesis of natural scrapie infection in sheep. APMIS 110:23–32. Vilotte JL, Soulier S, Essalmani R, Stinnakre MG, Vaiman D, Lepourry L, Da Silva JC, Besnard N, Dawson M, Buschmann A, Groschup M, Petit S, Madelaine MF, Rakatobe S, Le Dur A, Vilette D, Laude H. 2001. Markedly increased susceptibility to natural sheep scrapie of transgenic mice expressing ovine prp. J Virol 75:5977–5984. Vrentas CE, Greenlee JJ, Baron T, Caramelli M, Czub S, Nicholson EM. 2013. Stability properties of PrPSc from cattle with experimental transmissible spongiform encephalopathies: use of a rapid whole homogenate, protease-free assay. BMC Vet Res 9:167. Wadsworth JD, Asante EA, Desbruslais M, Linehan JM, Joiner S, Gowland I, Welch J, Stone L, Lloyd SE, Hill AF, Brandner S, Collinge J. 2004. Human prion protein with valine 129 prevents expression of variant CJD phenotype. Science 306: 1793–1796. Wadsworth JD, Joiner S, Linehan JM, Balkema-Buschmann A, Spiropoulos J, Simmons MM, Griffiths PC, Groschup MH, Hope J, Brandner S, Asante EA, Collinge J. 2013. Atypical scrapie prions from sheep and lack of disease in transgenic mice overexpressing human prion protein. Emerg Infect Dis 19:1731–1739. Wells GA. 2007. BSE terminology. Vet Rec 161:244. Wells GA, Konold T, Arnold ME, Austin AR, Hawkins SA, Stack M, Simmons MM, Lee YH, Gavier-Widen D, Dawson M, Wilesmith JW. 2007. Bovine spongiform encephalopathy: the effect of oral exposure dose on attack rate and incubation period in cattle. J Gen Virol 88(Pt 4):1363–1373. Wells GA, Scott AC, Johnson CT, Gunning RF, Hancock RD, Jeffrey M, Dawson M, Bradley R. 1987. A novel progressive spongiform encephalopathy in cattle. Vet Rec 121:419–420. Wells GA, Spiropoulos J, Hawkins SA, Ryder SJ. 2005. Pathogenesis of experimental bovine spongiform encephalopathy: preclinical infectivity in tonsil and observations on the distribution of lingual tonsil in slaughtered cattle. Vet Rec 156:401–407. Wells GA, Wilesmith JW, McGill IS. 1991. Bovine spongiform encephalopathy: a neuropathological perspective. Brain Pathol 1:69–78.


Smith JD, Greenlee JJ, Hamir AN, Greenlee MH. 2009. Altered electroretinogram b-wave in a Suffolk sheep experimentally infected with scrapie. Vet Rec 165:179–181. Smith JD, Greenlee JJ, Hamir AN, Richt JA, Greenlee MH. 2009. Retinal function and morphology are altered in cattle infected with the prion disease transmissible mink encephalopathy. Vet Pathol 46:810–818. Smith JD, Greenlee JJ, Hamir AN, West Greenlee MH. 2008. Retinal cell types are differentially affected in sheep with scrapie. J Comp Pathol 138:12–22. Sohn HJ, Lee YH, Green RB, Spencer YI, Hawkins SA, Stack MJ, Konold T, Wells GA, Matthews D, Cho IS. 2009. Bone marrow infectivity in cattle exposed to the bovine spongiform encephalopathy agent. Vet Rec 164:272–273. Somerville RA, Ritchie LA. 1990. Differential glycosylation of the protein (PrP) forming scrapie-associated fibrils. J Gen Virol 71:833–839. Soto C. 2003. Unfolding the role of protein misfolding in neurodegenerative diseases. Nat Rev Neurosci 4:49–60. Soto C, Satani N. 2011. The intricate mechanisms of neurodegeneration in prion diseases. Trends Mol Med 17:14–24. Spiropoulos J, Hawkins SA, Simmons MM, Bellworthy SJ. 2014. Evidence of in utero transmission of classical scrapie in sheep. J Virol 88:4591–4594. Stack MJ, Chaplin MJ, Clark J. 2002. Differentiation of prion protein glycoforms from naturally occurring sheep scrapie, sheeppassaged scrapie strains (CH1641 and SSBP1), bovine spongiform encephalopathy (BSE) cases and Romney and Cheviot breed sheep experimentally inoculated with BSE using two monoclonal antibodies. Acta Neuropathol 104:279–286. Sugiura K, Onodera T, Bradley R. 2009. Epidemiological features of the bovine spongiform encephalopathy epidemic in Japan. Rev Sci Tech 28:945–956. Terry LA, Marsh S, Ryder SJ, Hawkins SA, Wells GA, Spencer YI. 2003. Detection of disease-specific PrP in the distal ileum of cattle exposed orally to the agent of bovine spongiform encephalopathy. Vet Rec 152:387–392. Tester S, Juillerat V, Doherr MG, Haase B, Polak M, Ehrensperger F, Leeb T, Zurbriggen A, Seuberlich T. 2009. Biochemical typing of pathological prion protein in aging cattle with BSE. Virol J 6:64. Torres JM, Andreoletti O, Lacroux C, Prieto I, Lorenzo P, Larska M, Baron T, Espinosa JC. 2011. Classical bovine spongiform encephalopathy by transmission of H-type prion in homologous prion protein context. Emerg Infect Dis 17:1636–1644. Touzeau S, Chase-Topping ME, Matthews L, Lajous D, Eychenne F, Hunter N, Foster JD, Simm G, Elsen JM, Woolhouse ME. 2006. Modelling the spread of scrapie in a sheep flock: evidence for increased transmission during lambing seasons. Arch Virol 151:735–751. Tranulis MA, Benestad SL, Baron T, Kretzschmar H. 2011. Atypical prion diseases in humans and animals. Top Curr Chem 305:23–50. U.S. Department of Agriculture Animal and Plant Health Inspection Service. 2012. Summary Report: California Bovine Spongiform Encephalopathy Case Investigation [Internet]. Available online (http://www.aphis.usda.gov/animal_health/ animal_diseases/bse/downloads/BSE_Summary_Report.pdf ), accessed on March 10, 2015. U.S. Department of Agriculture Animal and Plant Health Inspection Service. 2015. National scrapie eradication program [Internet]. Available online (http://www.aphis.usda.gov/wps/ portal/aphis/ourfocus/animalhealth/sa_animal_disease_ information/!ut/p/a1/jY_LDoIwEEW_hQ8wHSvyWPKm


Williams ES. 2002. The transmissible spongiform encephalopathies: disease risks for North America. Vet Clin North Am Food Anim Pract 18:461–473. Williams ES. 2005. Chronic wasting disease. Vet Pathol 42:530–549. Williams ES, Young S. 1980. Chronic wasting disease of captive mule deer: a spongiform encephalopathy. J Wildl Dis 16:89–98. Wilson R, Plinston C, Hunter N, Casalone C, Corona C, Tagliavini F, Suardi S, Ruggerone M, Moda F, Graziano S, Sbriccoli M, Cardone F, Pocchiari M, Ingrosso L, Baron T, Richt J, Andreoletti O, Simmons M, Lockey R, Manson JC, Barron RM. 2012. Chronic wasting disease and atypical forms of bovine spongiform encephalopathy and scrapie are not transmissible to mice expressing wild-type levels of human prion protein. J Gen Virol 93(Pt 7):1624–1629. Wood JN, Done SH, Pritchard GC, Wooldridge MJ. 1992. Natural scrapie in goats: case histories and clinical signs. Vet Rec 131:66–68. Yamakawa Y, Hagiwara K, Nohtomi K, Nakamura Y, Nishijima M, Higuchi Y, Sato Y, Sata T. 2003. Atypical proteinase K-resistant prion protein PrPres observed in an apparently healthy 23month-old Holstein steer. Jpn J Infect Dis 56:221–222. Zlotnik I, Barlow RM. 1967. The transmission of a specific encephalopathy of mink to the goat. Vet Rec 81:55–56.


West Greenlee MH, Smith JD, Platt EM, Juarez JR, Timms LL, Greenlee JJ. 2015. Changes in retinal function and morphology are early clinical signs of disease in cattle with bovine spongiform encephalopathy. PLoS One 10:e0119431. Wilesmith JW. 1988. Bovine spongiform encephalopathy. Vet Rec 122:614. Wilesmith JW, Ryan JB, Atkinson MJ. 1991. Bovine spongiform encephalopathy: epidemiological studies on the origin. Vet Rec 128:199–203. Wilesmith JW, Wells GA, Cranwell MP, Ryan JB. 1988. Bovine spongiform encephalopathy: epidemiological studies. Vet Rec 123:638–644. Wilesmith JW, Wells GA, Ryan JB, Gavier-Widen D, Simmons MM. 1997. A cohort study to examine maternally-associated risk factors for bovine spongiform encephalopathy. Vet Rec 141:239–243. Will RG. 2003. Acquired prion disease: iatrogenic CJD, variant CJD, kuru. Br Med Bull 66:255–265. Will RG, Ironside JW, Zeidler M, Cousens SN, Estibeiro K, Alperovitch A, Poser S, Pocchiari M, Hofman A, Smith PG. 1996. A new variant of Creutzfeldt-Jakob disease in the UK. Lancet 347:921–925.

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Progress on low susceptibility mechanisms of transmissible spongiform encephalopathies.

From slow virus to prion: a review of transmissible spongiform encephalopathies.

Spongiform encephalopathies. The prion's progress.

Spongiform encephalopathies. PrP and the scrapie agent.

Abnormalities in Brainstem Auditory Evoked Potentials in Sheep with Transmissible Spongiform Encephalopathies and Lack of a Clear Pathological Relationship.

The human spongiform encephalopathies: kuru, Creutzfeldt-Jakob disease, and the Gerstmann-Sträussler-Scheinker syndrome.

Public health issues related to animal and human spongiform encephalopathies: memorandum from a WHO meeting.

Relationship of protease-resistant protein, scrapie-associated fibrils and tubulofilamentous particles to the agent of spongiform encephalopathies.

Could intracrine biology play a role in the pathogenesis of transmissable spongiform encephalopathies, Alzheimer's disease and other neurodegenerative diseases?

Cross-seeding of prions by aggregated α-synuclein leads to transmissible spongiform encephalopathy.

Assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay.

Autoimmune encephalopathies.

The age-dependent epileptic encephalopathies.

Detection and partial discrimination of atypical and classical bovine spongiform encephalopathies in cattle and primates using real-time quaking-induced conversion assay.

Paraneoplastic and autoimmune encephalopathies.

Bovine spongiform encephalopathy.

Toxic and metabolic encephalopathies: iconographic essay.

Delusional bovine spongiform encephalopathy.

Bovine spongiform encephalopathy.

Bovine spongiform encephalopathy.

Bovine spongiform encephalopathy.

Livestock metabolomics and the livestock metabolome: A systematic review.

Surgical treatment of pediatric epileptic encephalopathies.

Epileptic Encephalopathies in Childhood: The Role of Genetic Testing.