Differential Accumulation of Misfolded Prion Strains in Natural Hosts of Prion Diseases

Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are a group of neurodegenerative protein misfolding diseases that invariably cause death. TSEs occur when the endogenous cellular prion protein (PrPC) misfolds to form the pathological prion protein (PrPSc), which templates further conversion of PrPC to PrPSc, accumulates, and initiates a cascade of pathologic processes in cells and tissues. Different strains of prion disease within a species are thought to arise from the differential misfolding of the prion protein and have different clinical phenotypes. Different strains of prion disease may also result in differential accumulation of PrPSc in brain regions and tissues of natural hosts. Here, we review differential accumulation that occurs in the retinal ganglion cells, cerebellar cortex and white matter, and plexuses of the enteric nervous system in cattle with bovine spongiform encephalopathy, sheep and goats with scrapie, cervids with chronic wasting disease, and humans with prion diseases. By characterizing TSEs in their natural host, we can better understand the pathogenesis of different prion strains. This information is valuable in the pursuit of evaluating and discovering potential biomarkers and therapeutics for prion diseases.

A given prion disease within a species can have a range of phenotypes. Different phenotypes include, but are not limited to, differences in transmissibility within a species [19]; transmissibility between species [19]; disease time course [20]; most prominent clinical signs [21]; tissue, brain region [22] and cellular localization [23] of accumulated PrP Sc .
Often, disease phenotypes within a given species are denoted as different 'strains' [24]. Strains in a natural host are commonly defined by the molecular weight profile of the PrP Sc [25], genotypes of susceptible hosts [23], age of disease onset [26,27], and distribution and intensity of misfolded PrP Sc [20,28]. Because the molecular profile on a Western blot may be different depending on host species or strain of infection, to follow is a brief discussion of the significance. Following proteinase-K digestion of a sample homogenate discussion of the significance. Following proteinase-K digestion of a sample homogenate containing misfolded prion protein, a Western blot results in three bands that are the di-, mono-, and unglycosylated isoforms (highest kDa to lowest kDa, respectively) of the prion polypeptide. Increased glycosylation adds weight, which slows migration of the prion protein. Thus, the diglycosylated band has the highest kDa, while the unglycosylated has the lowest kDa. Different strains of prion disease in a given species may have different molecular weight profiles, particularly of the unglycosylated band, due to the differential cleavage by proteinase-K of the misfolded protein's n-terminal (reviewed in [29]).
Strains also may be grouped into what are referred to as 'classical' and 'atypical' based on proteinase K-resistant fragments, neuronal tropism, deposition patterns, and pathological lesion profile [30]. Classical cases of prion diseases tend to occur in groups of younger animals compared to atypical cases that tend to occur in individual older animals. Additionally, animals with classical cases of scrapie shed prions into the environment and thereby have a propensity for vertical and horizontal transmission under field conditions. Atypical cases of scrapie shed little to no prions into the environment, providing further evidence to support the spontaneous origin of atypical prion diseases. This, however, does not hold true in cases of cattle in which prions are not shed into the environment regardless of strain.
Rodent models have proven to be invaluable in isolating [31], identifying, and characterizing [32] strains of TSEs isolated from natural hosts [33]. However, to fully understand the pathogenesis of different TSE strains, they must also be characterized in their natural host. In many instances the character of PrP Sc immunoreactivity as well as cellular and subcellular localization of accumulated PrP Sc also differs between prion strains [34][35][36]. While Western blots provide information regarding protein size and strain following proteinase-K digestion, this review focuses on the differential localization and patterns of PrP Sc that are observed using immunohistochemistry. In this review, we focus on PrP Sc accumulation in three locations in the nervous system (retinal ganglion cells, cerebellar cortex, and enteric nervous system; see Figure 1). These three nervous system sites were selected based on the presence of robust literature that reports strain-dependent differential accumulation of PrP Sc within these structures. We discuss their utility in differentiating strains and better understanding the pathogenesis of bovine spongiform encephalopathy in cattle, scrapie in sheep and goats, and chronic wasting disease in cervids.

Bovine Spongiform Encephalopathy
Bovine spongiform encephalopathy (BSE) was first described in cattle in 1986 [37]. BSE is grouped into classical and atypical strains [38]. After Western blotting, the molecular weight profile of the unglycosylated band in atypical strains either lower (L-BSE) or higher (H-BSE) than the classical strain (C-BSE). C-BSE is transmissible to cattle [8], sheep [39], goats [39], and humans [40,41] among other animals [18] via consumption of infectious material and is the agent responsible for the mid-1980s to mid-1990s epizootic disease in the United Kingdom where over 178,000 cattle were diagnosed positive for C-BSE [42,43] and millions of cattle were depopulated. As of 2021, 232 people have died from vCJD worldwide [44]. While C-BSE is infectious following ingestion, all known atypical BSE strains are either spontaneous [38] or are inherited via polymorphism in the PRNP gene [45]. Atypical strains of BSE and scrapie have been identified in older animals [46] and do not appear to be easily transmitted via the oral route [47]. While atypical scrapie and H-BSE do not seem to be infectious to humans [48,49], evidence suggests that L-BSE has the potential to be transmissible to humans [49]. Relative to other species with prion diseases, cattle with BSE generally have little to no lymphoid distribution of PrP Sc , except for tonsils and transiently in Peyer's patches [50,51]. Though atypical strains appear in older animals, experimental intracranial transmission of atypical strains of BSE results in a much shorter incubation time compared to C-BSE [52,53]. Unlike other species, polymorphisms in the PRNP gene of cattle are extremely rare, thus disease susceptibility and incubation time is not affected by an animal's genotype [53].

Scrapie
Scrapie is the prion disease of sheep and goats. The first records of scrapie date back to 1732 [54]. Following the first description of atypical (Nor-98) scrapie in 2003, discussion of scrapie strains is typically framed using classical and atypical scrapie in sheep [3]. There are differences in the molecular weight profile between classical and atypical strains of scrapie, as atypical scrapie has a smaller PrP Sc fragment, is more PK sensitive, and has 5 less intense Western blot bands compared to the three strong bands in classical scrapie [3,4,26]. In sheep, classical scrapie is spread primarily via horizontal transmission shortly after animals are born, though the average age of affected animals is 2-5 years of age [55], and there is widespread distribution of PrP Sc in lymphoid tissues [56,57]. Atypical scrapie in sheep appears to be sporadic, in that new cases appear in isolation, as opposed to a cluster of infected animals [3], and there is little to no accumulation of PrP Sc in the lymphoid system [4,58]. There are a number of polymorphisms in the PRNP gene of sheep that affect susceptibility and incubation time of different strains of scrapie (reviewed in [26,59]). Experimental and epidemiological evidence suggests that there is an extremely low likelihood that scrapie could be transmitted to humans [29,60]. Still, studies show that this low likelihood leaves room for the possibility of scrapie transmission to humans [61,62].

Chronic Wasting Disease
Chronic wasting disease (CWD) is the prion disease of deer and elk [63]. CWD was first identified in 1967 and was formally described in 1980 in captive mule deer and blacktailed deer [64]. Several CWD strains have been described [31,[65][66][67][68]. In cervids with CWD, there is widespread accumulation of PrP Sc in lymphoid tissues [69]. There are several described polymorphisms in the PRNP gene that may influence disease susceptibility and incubation time [70]. CWD is extremely contagious between cervids, and while there is some evidence it is transmissible to other species [71][72][73][74], there is no evidence that it has been transmitted to humans [9,75].

Human Prion Diseases
The most common prion disease in humans is Creutzfeldt-Jakob disease (CJD). CJD consists of multiple strains that include iatrogenic, variant, familial, and sporadic CJD Viruses 2021, 13, 2453 4 of 31 (iCJD, vCJD, fCJD, and sCJD, respectively). The basis of these strains is their origin: acquired, inherited, or spontaneous [76]. For example, iCJD and vCJD are acquired. The cause of iatrogenic transmission of CJD is a surgical or medical procedure involving materials (pituitary hormones, dura mater graft, blood transfusion, etc.) contaminated with PrP Sc [76], while the variant strain is due to the ingestion of the misfolded prion protein from contaminated beef products [76]. Familial CJD is heritable and is the result of mutations at codons in the PRNP gene [76]. Lastly, sporadic CJD is spontaneous [76].
In 1974 the first case of iCJD was reported. This case was the first of many in which patients underwent a medical procedure with contaminated tissues or instruments and an incubation period of years to decades would follow. Due to subsequent public health and decontamination measures, iCJD does not pose the threat that it once did [77]. The first case of the vCJD epidemic in the United Kingdom was diagnosed in 1996, and evidence supports its cause being the consumption of cattle that were infected with BSE [40,41]. Susceptibility to vCJD is influenced by polymorphisms at codon 129 of the PRNP gene. For example, almost all vCJD cases are homozygous for methionine at codon 129 (MM129) [78][79][80]. Depending on the source, only one or two heterozygous individuals (MV129) have been reported to have vCJD [78,81,82], and no one homozygous for valine has reported to have acquired vCJD [78,82].
Prion diseases in humans can be caused by inherited mutations in the PRNP gene, the most common mutation causing familial CJD is E200K. The number (200) refers to the codon of the PRNP at which a mutation occurs. The letters (E and K) refer to the oneletter code for amino acids (glutamic acid and lysine). In the case of E200K, the resulting amino acid is E (glutamic acid), rather than K (lysine), at codon 200 of the PRNP gene. Occasionally, E200K-129M may be used. This indicates that the individual is homozygous for M (methionine) at codon 129 of the PRNP gene. Familial CJD is an inherited form of prion disease caused by mutations in the PRNP gene. As mentioned, the most common mutation is E200K [83], which is homologous [45,53] to the polymorphism observed in cattle with the hereditable E211K H-BSE [45]. At least 14 other autosomal dominant mutations causing fCJD have been reported [84].
Sporadic CJD has the greatest phenotypic variety and is genotype-dependent, as the majority of sCJD cases are homozygous for methionine (MM) at codon 129 of the PRNP gene [78]. The genotype of codon 129 of the PRNP gene and type of prion protein accumulation underlies the differentiation of subtypes of sCJD [85][86][87][88]. At codon 129 of the PRNP gene, people can be either heterozygous for methionine and valine (MV129) or homozygous for either methionine (MM129) or valine (VV129). Further, there are two types of prion accumulation that are designated Type 1 and 2. Type 1 prion accumulation has a PK cleavage site at residue 82 [86], a PK-resistant core of 21 kDa [85], and has a smaller aggregation size following filtration (mean pore size 72 nm) than Type 2 prion aggregation size [87]. Type 2 has a PK cleavage site at residue 97 [86], a PK-resistant core of 19 kDa [85], and has a larger aggregation size following filtration (mean pore size 72 nm) than Type 1 prion aggregation size [87]. In all, cases of sCJD can be broken down into the following pure subtypes based on genotype at codon 129 of the PRNP gene and type of prion accumulation: MM1, MV1, VV1, MM2, MV2, and VV2 [88][89][90]. Concurrent Type 1 and Type 2 prion accumulation occurs in over one-third of sCJD cases [88]. Prion strains can be differentiated by ratio of di-, mono-, and unglycosylated protein as well as relative molecular mass [91]. Interestingly, variants of sCJD can also present with visual disturbances, as people the Heidenhain variant of sCJD present with visual symptoms at disease onset along with early posterior cortical involvement [92].
Humans are susceptible to other prion diseases, such as Kuru, fatal familial insomnia (FFI), and Gerstmann-Straussler-Scheinker (GSS). Kuru, similar to vCJD, is an acquired prion disease. It is specific to the Fore people in New Guinea whose members participated in ritual cannibalism as a means of mourning and respect for their deceased kindred [93]. In the Fore language Kuru means trembling or shivering, which reflects the symptom onset of cerebellar ataxia and tremor [93]. Prevalence of the disease has dramatically decreased Viruses 2021, 13, 2453 5 of 31 due to the termination of the practice [93]. FFI, similar to fCJD, is a hereditary prion disease due to a mutation at codon 178 of the PRNP gene that substitutes asparagine with aspartic acid (D178N) and is autosomal dominant [94]. Lastly, GSS is also a hereditary prion disease and can be caused by several mutations. These mutations include P102L (most common) [83], P105L, A117V, F198S, Q217R, Q212P, and D202N [95]. Mutations causing GSS are autosomal dominant. It has been reported that people with GSS may have symptomatic abnormal eye movements and optic atrophy, the latter of which is extremely rare in people with CJD [96][97][98][99].
The eye is the most accessible part of the central nervous system. Thus, it can be imaged and functionally assessed using noninvasive techniques. Within the eye, the retina is responsible for transmitting visual stimuli to the brain for processing [138]. The retina is organized into functionally distinct cellular and synaptic layers of cells. PrP Sc differentially accumulates the retinal layers of animals with TSEs depending on factors ranging from strain of TSE to host genotype [52,53,100,119,125,136,139,140]. Once photons of light are transduced by the photoreceptor cells in the outer nuclear layer, the signal is transmitted to the inner nuclear layer and then the ganglion cell layer via the inner and outer plexiform layers. It is the axons of the retinal ganglion cells in the ganglion cell layer that exit the globe as the optic nerve to connect with subsequent parts of the visual pathway. Animals with TSEs accumulate PrP Sc in the retina and multiple parts of the visual pathway [109,125,141]. Within the cellular and synaptic layers of the retina, PrP Sc accumulation is most common in the plexiform layers of the retina [115,120,121,126,139,142,143] while the retinal ganglion cells demonstrate a strain-dependent variation of PrP Sc accumulation [52,53,58,100,125].
There are strain-dependent differences in retinal function, prion accumulation, glial response, autophagic response, neuroinflammation, and other morphologic changes in the retinas of animals with TSEs [52,100,110]. Electroretinograms and optical coherence tomography have been used to assess functional and morphologic differences between strains of prion disease in cattle [100], sheep [109], and goats [122]. While electroretinograms measure the retina's response to visual stimuli, optical coherence tomography can be used to measure retinal thickness. The earliest functional difference associated with the accumulation of pathological prion protein is present in electroretinogram data 11 months prior to clinical signs of disease in cattle with BSE [100]. Strain-dependent functional differences include prolonged b-wave implicit time [53,100,110,144] beginning at 12 months post inoculation [100] and continuing until clinical endpoint in cattle with C-BSE and H-BSE [100]. Additional data from optical coherence tomography shows retinal thinning in cattle [53,100,110] that is first detectable at 12 months post inoculation in cattle inoculated with C-BSE and H-BSE [100] and continues until clinical endpoint [100,110]. Strains of BSE can also be differentiated by the types of immunostaining for PrP Sc . In sheep, functional differences are evident by electroretinogram in the reduction of a-wave and b-wave amplitudes [109]. While not statistically significant, the average retinal thickness measured using optical coherence tomography in goats with scrapie trended lower than their scrapiefree counterparts [122]. PrP Sc accumulates in the retina of cervids with CWD; however, functional data are not available [121,125,127]. Electroretinogram data demonstrates that people with CJD have a significant reduction in b-wave amplitude [145,146]. Here we

Bovine Spongiform Encephalopathy
Generally, retinal tissues from cattle experimentally inoculated with C-BSE have less immunoreactivity for PrP Sc as compared to H-BSE, E211K H-BSE, and L-BSE [52,53,100,147]. These strain-dependent differences in the extent and intensity of PrP Sc immunoreactivity in the retinas of cattle are illustrated and summarized in Figure 2A-D and Table 1. The deposition pattern in the retinas of cattle inoculated with C-BSE has been described as ranging from punctate and granular [52] to multifocal coalescing granular and globular [100] staining that is localized to the plexiform layers. Retinal tissues from cattle with atypical BSEs have more PrP Sc immunoreactivity compared to C-BSE [38,52]. PrP Sc immunoreactivity is more extensive, intense, and uniform in the retinas of cattle with atypical BSEs compared to cattle with C-BSE, especially in the plexiform layers [52,100]. While cattle with C-BSE have little to no PrP Sc immunoreactivity outside the plexiform layers, cattle with H-BSE have staining in all retinal layers. In addition to globular deposits of misfolded prion protein in the photoreceptor cells, it also is reported that there are multifocal globular deposits in the nuclear layers of the retinas of cattle with H-BSE [52,100]. Cattle with H-BSE have intense granular PrP Sc accumulation in the plexiform layers [112]. Further differentiation between C-BSE and H-BSE can be conducted using the 12B2 antibody that is immunoreactive to tissues affected by H-BSE and not C-BSE [100]. Using the 12B2 antibody, tissues from cattle with C-BSE cannot be differentiated from control animal tissues while those with H-BSE displayed immunoreactivity throughout the retina [100]. The deposition pattern of PrP Sc in retinal ganglion cells of cattle experimentally inoculated with H-BSE is similar to that of cattle inoculated with E211K H-BSE, both demonstrating prominent intracellular accumulation [53]. Deposition of PrP Sc is intense and abundant in the plexiform layers in cattle inoculated with E211K H-BSE ( Figure 2D) [53,110]. In one study using three isolates of L-BSE (French low-type, Canadian low-type, and Italian BASE), no differences in PrP Sc deposition in the retina were reported between these isolates of L-BSE [52,148]. Similar to H-BSE, the deposition pattern in cattle experimentally inoculated with L-BSE is greater in amount and distribution compared to cattle inoculated with C-BSE [38,52]. Similar to H-BSE, the retinas affected by L-BSE have intense punctate deposition that is reported to be more intense and uniform in the plexiform layers compared to those of C-BSE [52,100]. Compared to cattle with H-BSE, the retinal layers of cattle with L-BSE have fewer deposits of PrP Sc ; however, these deposits are still greater than cattle with C-BSE [52]. Cattle with L-BSE demonstrate intense punctate PrP Sc accumulation in the plexiform layers of the retina [144]. mentally inoculated with L-BSE is greater in amount and distribution compared to cattle inoculated with C-BSE [38,52]. Similar to H-BSE, the retinas affected by L-BSE have intense punctate deposition that is reported to be more intense and uniform in the plexiform layers compared to those of C-BSE [52,100]. Compared to cattle with H-BSE, the retinal layers of cattle with L-BSE have fewer deposits of PrP Sc ; however, these deposits are still greater than cattle with C-BSE [52]. Cattle with L-BSE demonstrate intense punctate PrP Sc accumulation in the plexiform layers of the retina [144].  The greatest difference in staining between strains of BSE can be observed in the retinal ganglion cells. Intraneuronal staining of the retinal ganglion cells in cases of cattle with C-BSE is less than atypical cases [52,53,100]. Cattle with H-BSE have intense granular PrP Sc accumulation in the ganglion cell layer [112] along with hallmark intense globular [100] and robust intracellular [52] PrP Sc deposits present in the retinal ganglion cells ( Figure 2C) [52,100]. Deposition of PrP Sc is a prominent feature in the cell bodies of the retinal ganglion cells in cattle inoculated with E211K H-BSE ( Figure 2D) [53,110]. Importantly, the retinal ganglion cells of cattle with L-BSE have intense punctate [144] and robust intracellular [52] deposits of misfolded prion protein ( Figure 2B). Overall, PrP Sc accumulation is differential in the cell bodies of retinal ganglion cells of cattle with atypical BSEs that is increased compared to cattle with C-BSE.

Scrapie
There is differential accumulation of misfolded prion protein in the retinas of sheep with classical and atypical scrapie. These differences are best demonstrated in Figure 2E-H and Table 2. Of the two US isolates of classical scrapie in sheep, No. 13-7 and x124 [59,115,149], no differences were reported in the PrP Sc deposition patterns between retinas [115]. In sheep oronasally inoculated with the No. 13-7 classical strain, sheep homozygous for lysine at codon 171 (KK171) of the PRNP gene were resistant, while sheep with glutamine at codon 171 (QK171 or QQ171) accumulated PrP Sc in the retina [119]. The plexiform layers of the retinas in sheep with classical scrapie have confluent, punctate, globular, intense, and coarse particulate deposition patterns [109,[113][114][115][116]118,124]. In sheep with classical scrapie, the photoreceptor cell layer has multifocal punctate [114,115,118] deposits with granules in the inner segments of the photoreceptor cell layer [109,113,116,150]. Interestingly, sheep of AA136 genotype and oronasally inoculated with x124 classical scrapie were resistant while those intracranially inoculated accumulated PrP Sc in the retina [115]. The retinas of the No. 13-7-inoculated counterparts of this study all had immunoreactivity for PrP Sc [115]. Evidence suggests that PrP Sc accumulates differently in the retinas of sheep with atypical scrapie compared to those with classical scrapie [58] although the retina is not otherwise reported. In the retinas of sheep with atypical scrapie there is prominent immunoreactivity for PrP Sc in the plexiform layers with minimal staining in the nuclear layers ( Figure 2H) [58]. Results of immunohistochemical staining of the retina were not reported in other strains of scrapie such as CH1641 and SSBP1 [151]. In the retinas of goats with classical scrapie, the PrP Sc deposition pattern reflects what is observed in sheep with classical scrapie [122,123]. PrP Sc accumulation is intense and extensive the goats with classical scrapie [122,123]. Along these lines, there is strong [123] particulate [122] accumulation in the plexiform layers and weak accumulation in the nuclear layers [123].
Retinal tissues from sheep with classical scrapie have PrP Sc immunoreactivity in the cell bodies of retinal ganglion cells while RGC immunostaining negligible in sheep with atypical scrapie. The ganglion cell layer in sheep with classical scrapie has multifocal punctate deposits of PrP Sc [109,[113][114][115][116]118] with granular [109,124] and coarse particulate [116,124] deposits occurring in the cell bodies of retinal ganglion cells [113] ( Figure 2E-G). In the retinas of sheep with atypical scrapie, there is minimal staining in the ganglion cell layer ( Figure 2H) [58]. In goats with classical scrapie, there is intense accumulation in the ganglion cell layer [122] and strong prominent cytoplasmic deposition in retinal ganglion cells [123]. Similarly, intense intraneuronal PrP Sc accumulation is reported in the retinal ganglion cells of goats with classical scrapie ( Figure 2G) [122,123]. In summary, increased PrP Sc accumulation occurs in the retinal ganglion cells of sheep and goats with classical scrapie and not in those with atypical scrapie.

Chronic Wasting Disease
PrP Sc accumulates in the retinas of cervids with CWD [121,125,[127][128][129][130][131][132][133] and is different depending on the species affected by CWD and genotype within a species. For example, the degree of PrP Sc accumulation in the retinas of Rocky Mountain elk [125,130,132] is genotype-and strain-dependent based on the polymorphism at codon 132 of the PRNP gene [125,130]. These differences are best demonstrated in Figure 2I,J and Table 3. The most common genotype at codon 132 in captive and free-ranging Rocky Mountain elk is homozygous methionine (MM132) [125]. When elk of the MM132 genotype have CWD, their retinas display intense staining in the plexiform layers and no intraneuronal PrP Sc immunoreactivity in the retinal ganglion cells [125,130]. The retinas of Rocky Mountain elk heterozygous at codon 132 of the PRNP gene (ML132) demonstrate intracytoplasmic accumulation of PrP Sc [125]. Rocky Mountain elk homozygous for leucine at the codon 132 of the PRNP gene (LL132) have heavy intraneuronal PrP Sc accumulation in the cell bodies of retinal ganglion cells compared to MM132 ( Figure 2I,J) [130]. Passage of the CWD agent through LL132 elk may result in a strain separate than what is isolated from ML132 and MM132 elk [31]. Overall, retinal tissues from Rocky Mountain elk with the LL132 strain have more extensive PrP Sc immunoreactivity when compared to other genotypes and strains.
Other cervids with CWD also accumulate PrP Sc in the retina. Misfolded prion protein accumulates in the retinas of white-tailed deer [127][128][129]133], reindeer [131,132], and mule deer [121]. Within each species, no differential PrP Sc accumulation has been reported between retinas in a genotype-or strain-dependent manner. Reindeer accumulate PrP Sc in the retina [131,132] with intraneuronal immunoreactivity in the retinal ganglion cells [132]. Immunoreactivity for PrP Sc in the retinas of reindeer can be punctate, particulate, and coalescing deposits in the plexiform layers with scattered intramicroglial deposits [132]. Mule deer accumulate PrP Sc in the retina [121]. While prion accumulation is reported in the plexiform and ganglion cell layers of mule deer [121], there has been no further characterization of this staining. Polymorphisms exist at codons 95 and 96 of the PRNP gene in white-tailed deer, but there were no differences in PrP Sc immunoreactivity based on genotype [127][128][129]133]. PrP Sc staining patterns in the retinas of white-tailed deer are diffuse granular in the plexiform layers [127,129] and fine multifocal in the ganglion cell layer with little to no accumulation in the retinal ganglion cells as seen in Figure 2K [128]. In contrast, white-tailed deer inoculated with the sheep scrapie agent have robust accumulation of PrP Sc in the cell bodies of retinal ganglion cells ( Figure 2L) [152]. This is notable because it provides a way to differentiate CWD from scrapie in white-tailed deer.

Human Prion Diseases
PrP Sc accumulation has been demonstrated in the retinas of people with CJD [134][135][136][137]. These differences are summarized in Table 4. In cases of the sporadic, variant, familial, and iatrogenic strains of CJD, PrP Sc accumulation occurs primarily as strong uniform staining in the plexiform layers [134][135][136]. In people with the most common subtype of sCJD (MM1), the plexiform layers are reported to be immunoreactive in a focal granular pattern [134]. There is no intraneuronal staining of the retinal ganglion cells reported in people with sCJD and vCJD [135,136]. PrP Sc accumulation is both genotype-and strain dependent because people with sCJD of the most common subtype (MM1) have less PrP Sc accumulation in the retina compared to both people with sCJD of a less common subtype and vCJD [134]. Supporting strain-dependent differential PrP Sc accumulation, people with vCJD have a higher concentration of PrP Sc in the retina relative to brain than people with sCJD [137]. The accumulation of PrP Sc in the retina has not been reported in studies of people with GSS [95,153,154] or Kuru [155][156][157][158].

Cerebellar Cortex and White Matter
Depending on the strain of TSE and host genotype, misfolded prion protein accumulates differentially in the cerebellar cortex of cattle with BSE [53,159,160], sheep and goats with scrapie [23,26,29,161], cervids with CWD [129,132,162], and humans with CJD, GSS, and Kuru [95,158,163]. These strain-dependent differences include the intensity of PrP Sc accumulation relative to the brainstem at the level of the obex [23,26,29,160,161], immunolabelling pattern [129,159,161], cerebellar cortex layer with greatest PrP Sc accumulation [26,129,159], and molecular profile [110]. Within the cerebellum, the cerebellar cortex consists of three cellular layers: the molecular layer, Purkinje cell layer, and granule cell layer. Immediately deep to the granule cell layer of the cerebellar cortex is the cerebellar white matter that allows axonal fibers to enter and exit the cerebellum for communication with the cerebrum and body.
The role of the cerebellar cortex is to receive and integrate information from the brain and body in order to produce coordinated goal-directed movements as well as maintain posture and balance. This role of the cerebellar cortex includes correcting errors in voluntary movement via receiving signals from the cerebrum and feedback from the body, which it can then integrate into appropriate efferent signals to ultimately synapse with somatic muscle fibers. Lesions in the cerebellum manifest as errors in goal-directed movement and posture. For example, animals or people may present with uncoordinated flexor and extensor muscles, ataxia, or hypermetria. These manifestations of cerebellar dysfunction are evident clinical signs in animals and people with prion diseases [4,58,[164][165][166]. However, clinical signs do not always reflect the level of PrP Sc accumulation in the cerebellum. For example, goats with scrapie may accumulate a similar level of PrP Sc in the cerebellum compared to sheep; however, they may not present with cerebellar signs [164]. Still, the molecular and granule cell layers of the cerebellar cortex display differential accumulation of misfolded prion protein. Here, when discussing the intensity of misfolded prion accumulation in the cerebellum, it is always relative to the brainstem at the level of the obex. We summarize the differential accumulation that occurs in the cerebellar cortex of animal and humans with prion disease (Tables 5-9).        No intraneuronal staining (Ferrer 2000) Coarse dotted deposits (Yang 1999

Bovine Spongiform Encephalopathy
There are differences in the intensity and patterns of PrP Sc accumulation in the cerebella of cattle with different strains of BSE [47,53,110,112,159,160,[167][168][169]. A summary of these differences can be found in Table 5. In cattle with C-BSE, there is more PrP Sc deposition in the molecular layer than in the granule cell layer [53]. In the molecular layer there is prominent stellate and linear staining patterns whereas the granule cell layer is made up of fine and coarse granular to aggregated staining patterns [53] [53,167,168,170]. In relation to the brainstem at the level of the obex, the cerebellar cortices of cattle with L-BSE have significantly stronger PrP Sc signal intensities [47,160] than cattle with C-BSE and H-BSE [160]. Cattle with L-BSE have an even distribution of immunoreactivity in the molecular and granule cell layers [159]. The PrP Sc accumulation in the cerebellum of cattle with L-BSE was diffuse and is a similar pattern to the accumulation observed in the cerebellar cortices of sheep with atypical scrapie [159]. Compared to cattle with L-BSE, the molecular and granule cell layers of cattle with H-BSE have substantially less PrP Sc accumulation that is less uniform [159]. Cattle with H-BSE have a stellate immunolabelling of PrP Sc with plaques in the cerebellar cortices [112] and pronounced widespread glial staining in the white matter of the cerebellum when compared to C-BSE and L-BSE counterparts [112,159]. In the E211K H-BSE, staining of the cerebellum is scant with small, multifocal clumps of PrP Sc in both the molecular and granule cell layers [110]. Further, there is a fine granular, particulate, and stellate immunolabelling pattern in the molecular layer with coarse granular and particulate labelling of PrP Sc in the granule cell layer [53]. Cattle with the E211K H-BSE generally do not display immunoreactivity in the cerebellar white matter, differing from cattle with H-BSE in which the most prominent PrP Sc staining occurs in the cerebellar white matter [110,159]. In cattle with E211K H-BSE, immunolabelling against PrP Sc in the granule cell layer is comparable to that of C-BSE. Cattle with E211K H-BSE have fine granular and stellate labelling in the molecular layer of the cerebellum while cattle with C-BSE have prominent stellate and linear labelling in the molecular layer [53]. Little information is specifically reported on the Purkinje cells of cattle with BSE; however, published images indicate that there is little to no staining of Purkinje cells in any strain of BSE [53,159]. Overall, the greatest difference in the cerebellar cortices of cattle with different strains of BSE is L-BSE in which there is a significantly greater concentration of PrP Sc accumulation in relation to the brainstem at the level of the obex. These differences suggest that the cerebellum is more reliable by immunohistochemistry for differentiating between strains of BSE than the brainstem at the level of the obex in cattle with BSE [159].

Scrapie
The relative intensity and pattern of PrP Sc accumulation varies in the cerebella of sheep and goats with different strains of scrapie [4,26,58,119,122,123,161,164,171,172]. A summary of these differences can be found in Tables 6 and 7. Overall, PrP Sc deposition in the cerebellum (relative to the brainstem at the level of the obex in the same animal) is less intense and widespread in cases of classical scrapie compared to cases of atypical scrapie [26]. In sheep with classical scrapie, the cerebellum has strong immunoreactivity for PrP Sc that is stronger in the granule cell layer than the molecular layer [26]. The staining is multifocal in the molecular layer and white matter [26]. The Purkinje cells in sheep with classical scrapie have some intraneuronal PrP Sc accumulation [119]. The staining in the cerebella is genotype-dependent at codon 171 of the PRNP gene in sheep with classical scrapie [119]. Sheep with lysine at codon 171 of the PRNP gene (QK171 or KK171) had similar staining in the cerebella and was different from sheep homozygous for glutamine (QQ171) [119]. The former commonly have accumulation of misfolded prion protein in the white matter while the latter sheep lack this PrP Sc accumulation [119]. Overall, the patterns of accumulation in the granule cell and molecular layer in the cerebellar cortices in classical scrapie is not genotype-dependent at codon 171 of the PRNP gene: granular, intraneuronal, and intraglial in the granule cell layer as well as scant punctate, granular, and stellate patterns in the molecular layer [119].
In sheep with atypical scrapie, the cerebellum has intense immunoreactivity for PrP Sc that is stronger in the molecular layer than the granule cell layer [26,58,173]. Sheep with atypical scrapie primarily have PrP Sc accumulation in the cerebellar cortex [3,4,58,161,171,173] while sheep with classical scrapie do not [4,26]. In sheep with atypical scrapie the staining patterns in the cerebellum is granular and punctate [58,173]. Cases of atypical scrapie have varied staining in the cerebellar white matter [161]. Unlike cases of classical scrapie in sheep, the Purkinje cells of those with atypical scrapie are always negative [161]. Again, atypical cases of scrapie have higher relative amounts of misfolded prion accumulation in the cerebellum in relation to their brainstem compared to the same structures in sheep with classical scrapie [58]. Still, some diversity exists in the degree of PrP Sc deposition, as it has been reported that some sheep with atypical scrapie have cerebella that are minimally affected [171]. When classical scrapie presents in goats, there is PrP Sc accumulation in the cerebellum [122,123,164]. Subpial immunolabelling against PrP Sc occurs in the cerebella of goats with classical scrapie [122]. Cytoplasmic staining is strong and widespread in the molecular layer, granule cell layer, Purkinje cells, and cerebellar white matter [123]. In sheep with scrapie, the cerebellum allows for differentiation between strains. While the cerebellar cortex displays PrP Sc immunoreactivity in sheep with classical and atypical strains of scrapie, it is greater in sheep with atypical scrapie in relation to PrP Sc accumulation in the brainstem at the level of the obex. Additionally, sheep with classical scrapie tend to accumulate more misfolded prion protein in the granule cell layer while sheep with atypical scrapie accumulate more PrP Sc in the molecular layer.

Chronic Wasting Disease
The misfolded prion protein deposits differentially in both intensity and pattern in the cerebella of cervids with CWD, as species and genotype both affect PrP Sc accumulation [129,132,162,174]. A summary of these differences can be found in Table 8. The accumulation of misfolded prion protein differs in the cerebella of white-tailed deer based on genotype. In experimentally inoculated white-tailed deer, there are two PRNP codons associated with differences in PrP Sc accumulation. The white-tailed deer wild-type prion protein allele is Q95/G96; codon 95 can instead be histidine (H) and codon 96 can instead be serine (S). Differences in accumulation pattern and intensity of PrP Sc will be discussed in the following order: wildtype, S96, H95, H95/S96. In wildtype white-tailed deer, there is abundant coalescing PrP Sc and plaques stretching in the granule cell layer and Purkinje cell layer. This occurs as coarse granular and large plaques [129]. In deer of the S96 genotype, accumulation of PrP Sc is confined to the granule cell layer and white matter.
In comparison to wildtype white-tailed deer, deer of the S96 genotype display less intense accumulation that is granular and diffuse [129]. Even less PrP Sc accumulation occurs in the cerebellar cortices of deer of the H95 genotype that are infected with CWD [129] with the predominance of staining in the granule cell layer and minimal labelling in the Purkinje cell and molecular layers. This accumulation of PrP Sc is discontinuous and diffuse in the granule cell layer as fine punctate and coarse small granular deposits with some plaque-like deposits [129]. When these polymorphisms are simultaneous (H95/S96), accumulation of PrP Sc manifests as fine punctate and coarse granular deposits that were evenly distributed in the granule cell layer [129]. The molecular layer of white-tailed deer with H95/S96 polymorphisms demonstrated more intense accumulation of PrP Sc than the deer of the wildtype, S96, and H95 genotypes [129]. White-tailed deer of the H95/S96 genotype displayed stellate aggregates of misfolded prion protein in the molecular layer of the cerebellar cortex [129].
In reindeer, PrP Sc accumulation ranges in intensity overall [132], but is consistently more intense in granule cell layers when compared to the molecular layer [13,132] with Purkinje cells devoid of accumulation [132]. Reindeer show punctate deposits of PrP Sc throughout the cerebellar cortex in less intense cases while particulate and aggregated deposits occur in more intense cases [132]. The cerebellar cortex of Rocky Mountain elk affected by CWD has glial-associated PrP Sc accumulation that was granular to punctate and was most prominent in the white matter [130]. Mule deer with CWD have accumulation of PrP Sc plaques in the cerebellar cortex that are present in the granule cell layer and molecular layer [174]. While the cerebellar cortex offers discernable pathological phenotypes in white-tailed deer with CWD for potential strain differentiation, there is not enough characterization in other cervids to draw conclusions.

Human Prion Diseases
PrP Sc accumulates in the cerebella of humans with prion diseases [95,163,166,[175][176][177]. A summary of these differences can be found in Table 9. The immunostaining pattern in the cerebellar cortex of people with vCJD is described as diffuse and florid plaques [175]. The density of PrP Sc accumulation was increased in the granule cell layer compared to the molecular layer of the cerebellar cortices in people with vCJD [175]. One report on an individual with dura mater-derived iCJD states that there was Type 1 PrP Sc accumulated in the cerebellar cortex. A synaptic staining pattern was found in both the molecular and granule cell layer of the cerebellar cortex [177]. When PrP Sc accumulates in humans with sCJD, reports vary. Some state that people with sCJD accumulate PrP Sc equally in the molecular and granule cell layer of the cerebellum [178], whereas others report that coarse dotted deposits of PrP Sc accumulate in the granule cell layer and diffuse fine dotted deposits in the molecular layer [163,179]. There is evidence that staining patterns vary depending on the subtype of sCJD. People with either the MM1 or MV1 subtype display fine punctate deposits with diffuse aggregates that were occasionally described as coarse [85,89]. PrP Sc plaques in the cerebellum of people with VV1 or MM2 subtype are rare, although more common in people of the MM2 subtype [85,89]. Otherwise, PrP Sc deposition in people of the MM2 subtype is described as coarse [85]. People with the VV2 and MV2 subtype have prominent involvement of the cerebellum [85,89] with these subtypes displaying plaque-like deposits primarily in the granule cell layer [85]. The VV2 subtype does not display Kuru-like deposits while diffuse plaques and punctate deposits are common [89]. In people of the MV2 subtype, kuru-like plaques are prominent [89].
Staining patterns are clearly different between people with sCJD compared to people with GSS [163]. Patients with GSS have Kuru plaques with synaptic localization of PrP Sc in both the molecular and granule cell layer [163]. Amyloid deposits of PrP Sc occur in both the molecular and granule cell layers of the cerebellum in people with GSS [95,153] and increased deposition occurs in the molecular layer [165]. For those with Kuru, misfolded prion protein accumulates in the cerebellum [155,157,180]. In the cerebellar cortices of people with Kuru, PrP Sc is not prominent in the molecular layer and is greater in the Purkinje cell and granule cell layer as fine granular diffuse deposits and plaques [155,157,158].

Enteric Nervous System
In animals with prion diseases, PrP Sc accumulates differentially in the enteric nervous system, a division of the nervous system that spans the entire digestive tract. The differential accumulation of PrP Sc is influenced by route of infection in addition to being both strain-dependent and genotype-dependent. While the enteric nervous system has long been thought to be a portal of PrP Sc entry to the central nervous system, it was not definitively demonstrated until 1999 [181].
PrP Sc accumulates differentially in the enteric nervous system of animals with prion diseases and these differences may allow for greater understanding of TSE pathogenesis and strain differentiation. Accumulation is not always predictable by route of infection. Beyond strain, differences in PrP Sc accumulation in the enteric nervous system occur due to propagation differences between natural infection, experimental intracranial inoculation, and experimental oronasal inoculation. Positive accumulation in the enteric nervous system is the result of centrifugal spread throughout the host following oronasal inoculation versus centripetal spread following intracranial inoculation. For example, AA136 sheep that were oronasally inoculated with the scrapie strain x124 were not susceptible to the agent and therefore did not accumulate PrP Sc in the enteric nervous system. However, AA136 sheep that were intracranially inoculated with the x124 scrapie strain were positive for PrP Sc in the enteric nervous system. The No. 13-7 scrapie strain counterparts in this study displayed different results. The No. 13-7 AA136 oronasally inoculated sheep were positive in the enteric nervous system while the intracranially inoculated AA136 sheep were negative [115]. Further, enteric nervous system accumulation did not occur following oral inoculation of atypical L-BSE although the cattle in this study was positive in other peripheral tissues [47]. Interestingly, it has also been reported that peripheral tissues accumulated PrP Sc in cattle intracranially inoculated with BSE; however, the enteric nervous system remained negative [159,182]. In another study, cattle orally challenged with C-BSE demonstrated PrP Sc accumulation in the enteric nervous system throughout the entire time course [183]. In addition to being strain-dependent, the accumulation of misfolded prion protein is genotype-dependent [184]. Here we summarize the differential accumulation of PrP Sc in the myenteric (Auerbach's) plexus and submucosal (Meissner's) plexus throughout the enteric nervous system in cattle with BSE, sheep and goats with scrapie, cervids with CWD, and humans with prion diseases (Tables 10-14). We will specify inoculation route throughout.

Bovine Spongiform Encephalopathy
PrP Sc accumulates in the myenteric plexus and submucosal plexus of the enteric nervous system in cattle with C-BSE following oral inoculation and subsequent PrP Sc uptake [183,[185][186][187][188]. A summary of these differences can be found in Table 10. The staining pattern following oral inoculation in the myenteric and submucosal plexuses was not clustered and localized to positive lymph follicles. In these cases, it was suggested that this could be due to direct neuroinvasion that evades gut-associated lymphoid tissue following oral inoculation [186]. The earliest PrP Sc accumulation is observed in the myenteric plexus of cattle with C-BSE is at 16 months post-inoculation in the ileum during preclinical stages [186]. Immunolabelling against PrP Sc is observed to have limited involvement that lasts until clinical stages of the disease following oral inoculation; however, distribution of PrP Sc in the enteric nervous system was wider in cattle with longer incubation periods [186]. Intense staining occurs at end stages of C-BSE in orally inoculated cattle that manifests as linear, intraglial, intraneuronal, and perineuronal with an association to the satellite cell in the myenteric and submucosal plexuses of the enteric nervous system [183,186]. There was no obvious association between immunolabelling against PrP Sc in the enteric nervous system and positive Peyer's patch follicles [183,186]. H-BSE and L-BSE showed no enteric nervous system involvement, even following the successful oral transmission of atypical BSE [47,159]. Immunohistochemistry failed to demonstrate the presence of PrP Sc accumulation in the enteric nervous system of cattle with E211K H-BSE following intracranial inoculation [53,110].

Scrapie
PrP Sc accumulates differentially in the enteric nervous system of sheep with classical scrapie [181,[189][190][191][192], though this is not the case in sheep with atypical scrapie, as these sheep have no PrP Sc immunoreactivity outside the central nervous system [58]. A summary of these differences can be found in Tables 11-13. Studies often report the genotype of sheep as a sequence of three one-letter amino acid codes at codons 136, 154, and 171 of the PRNP gene that are of interest due to their influence on susceptibility to scrapie. This section reports genotypes in such a manner.
The distribution and intensity increase as classical scrapie progresses until the entire enteric nervous system has abundant PrP Sc deposition in clinical stages [189]. In sheep with classical scrapie, intraneuronal fine granules of PrP Sc deposit in the myenteric and submucosal plexuses [181]. Sheep expressing VRQ/VRQ at codons 136, 154, and 171 of the PRNP gene displayed more extensive PrP Sc accumulation in the enteric nervous system than less susceptible sheep. Sheep that expressed VRQ/VRQ were positive in all gastrointestinal sites while sheep that expressed VRQ/ARQ only showed PrP Sc accumulation from the forestomaches to the rectum; sheep that were ARQ/ARQ had PrP Sc accumulation in the enteric nervous system of the omasum, abomasum, and intestines (sans esophagus, reticulum or rumen) [181]. The earliest detection of PrP Sc accumulation in sheep with classical scrapie is at 5 months post inoculation [191]. Sheep with classical scrapie display strong intraneuronal labelling of the ganglion and satellite cells in both myenteric and submucosal plexuses along the large and small intestines [192]. A sparse amount of neurons in the abomasum and duodenum of sheep with classical scrapie had misfolded prion protein accumulation [192]. Unlike cattle, the deposition of PrP Sc in the enteric nervous system of sheep with classical scrapie parallels the extent of deposition in the lymphoreticular system of the gut. Intraneuronal PrP Sc was not in enteric ganglia of forestomaches [192]. PrP Sc accumulation in classical scrapie is genotype-dependent, as enteric neuron staining did not occur in ARR/ARR or ARR/ARQ sheep [192]. This disease-specific intraneuronal immunolabelling of PrP Sc did not occur in the ganglia of the enteric nervous system in the forestomaches of sheep with classical scrapie [192]. With a sensitive protocol, PrP Sc deposition was shown to occur in the ganglia of the myenteric plexus and submucosal plexus in addition to nerve fibers in the submucosa [192]. PrP Sc deposition did not occur in the enteric nervous system near lymphoid nodules associated with inflammatory foci [192] nor was observed in the myenteric plexus at sites apart from large lymphoid aggerates in the jejunum and ileum during points in the incubation period [190]. In the forestomaches of sheep naturally infected with classical scrapie following natural infection, the enteric nervous system of the abomasum is first positive in sheep 9 months old, in which PrP Sc accumulation is present in the autonomous myenteric nervous system (and central nervous system) [190]. This study reports localization near Peyer's patches [190]. Misfolded prion protein accumulates first in the ileum before progressively spreading to adjacent tissues [190]. In this time course study following natural infection, the most severe case at 9 months of age displayed differential accumulation of PrP Sc in the autonomic myenteric nervous plexus with concomitant accumulation in the medulla oblongata at the level of the obex, specifically in the parasympathetic nucleus of the vagus nerve, that match staining in clinical adult controls [190]. Accumulation of the misfolded prion protein in the autonomic myenteric plexus occurred following its deposition in lymphoid tissues [190].
Genotype affects susceptibility to scrapie infection; therefore, there is no PrP Sc accumulation in the enteric nervous system of sheep with resistant genotypes (ARK/ARK and ARQ/ARQ). For example, intracranially inoculated sheep homozygous for lysine at codon 171 of the PRNP gene (ARK/ARK) are negative for PrP Sc in the enteric nervous system, while those with glutamine (ARQ/ARK or ARQ/ARQ) are positive [119]. Additional support for genotype dependence occurs in a study comparing two classical scrapie strains: No. 13-7 and x124 after intracranial (IC) or intranasal (IN) inoculation [115]. Accumulation in sheep with No. 13-7 compared to x124 classical scrapie is both strain-dependent and genotype-dependent in the enteric nervous system. PrP Sc accumulates readily in all genotypes inoculated with No. 13-7, whereas accumulation is less so in sheep with x124 [115]. In sheep with x124 classical scrapie, the most susceptible sheep (VRQ/VRQ) were positive for PrP Sc while the least susceptible (ARQ/ARQ) were negative [115]. Of note, the enteric nervous system was positive in intracranially inoculated sheep with ARQ/ARQ genotype [115]. PrP Sc displayed as fine granules intraneuronally in enteric neurons and glial cells of sheep with classical scrapie as well as the cell membranes of neurons of both the myenteric and submucosal plexuses [115]. Immunolabelling of the foregut varied by strain and genotype [115]. The enteric nervous system of the reticulum had PrP Sc deposits (13-7 IC ARQ/ARQ), rumen and abomasum (13-7 IN VRQ/VRQ), and omasum (x124 IN VRQ/VRQ, 13-7 IN ARQ/VRQ) [115]. The PrP Sc deposition varied in the enteric nervous system of the jejunum, ileum, and cecum [115]. Immunolabelling in the enteric nervous system occurs at the same time as gut-associated lymphoid tissue in sheep with No. 13-7 classical scrapie, whereas concurrent accumulation in sheep with x124 only occurred in VRQ/VRQ sheep [115]. Interestingly, the enteric nervous system did not show PrP Sc accumulation in ARQ/ARQ sheep with oronasally inoculated x124, who were not susceptible to scrapie, while it was positive in ARQ/ARQ sheep with intracranially inoculated x124 [115].
The onset of PrP Sc accumulation in the enteric nervous system is genotype dependent in sheep with classical scrapie [184]. Sheep expressing VRQ/VRQ in the PRNP gene the enteric nervous system was positive in all sheep at 112 dpi [184]. The following is a list of genotypes in increasing dpi at onset of PrP Sc accumulation in the enteric nervous system in sheep with classical scrapie: VRQ/ARQ (177 dpi), ARQ/ARQ (220 dpi), VRQ/ARR (366 dpi) [184]. The longest time period it took for any sheep in a genotype cohort to present with PrP Sc in the enteric nervous system was 2252 dpi in sheep that are ARQ/ARR [184]. Overall, this indicates that sheep more susceptible genotypes accumulate PrP Sc earlier in the enteric nervous system than more resistant sheep [184].
Further, intraneuronal accumulation occurred in enteric glial cells [193]. Sheep homozygous for ARR/ARR one month following inoculation with classical scrapie did not show PrP Sc accumulation and other sheep in this cohort remained heathy at 1.5 years following inoculation [194]. Sheep with clinical classical scrapie displayed widespread PrP Sc deposition in the enteric nervous system [194]. In this study, only sheep with clinical scrapie had PrP Sc accumulation in the enteric nervous system while this did not occur in sheep at earlier time points. Some studies suggest that amplification in the lymphoreticular system occurs prior to accumulation of PrP Sc in the enteric nervous system of sheep with classical scrapie [189,191] while others suggest simultaneous exposure to infection [194]. In sheep with atypical scrapie, there is no deposition of PrP Sc in the enteric nervous system regardless of genotype [58,195]. In other reports of atypical scrapie in sheep, involvement of the enteric nervous system was not investigated [3,196]. In goats with classical scrapie, PrP Sc is reported in the myenteric and submucosal plexuses of the enteric nervous system [122,123].

Chronic Wasting Disease
PrP Sc accumulates in the myenteric and submucosal plexuses of the enteric nervous system in cervids with CWD [129,131,133,197,198]. A summary of these differences can be found in Table 14. In white-tailed deer that were orally inoculated with CWD, PrP Sc accumulation occurred in the nerve fibers and ganglia of the enteric nervous system throughout the intestine [129]. Distribution of PrP Sc accumulation was genotype-dependent in whitetailed deer with CWD [129]. White-tailed deer with the genotypes H95/G96 or H95/S96 had less PrP Sc accumulation in the villi and crypts of the intestinal mucosa than whitetailed deer with the genotypes Q95/G96 or Q95/S96 deer [129]. This difference happened to the greatest degree at the ileocecal junction, as Q95/G96 and Q95/S96 white-tailed deer displayed strong PrP Sc accumulation, whereas H95/G96 and H95/S96 deer showed no PrP Sc accumulation [129]. In Rocky Mountain elk naturally infected with CWD, PrP Sc accumulation in the enteric nervous system was evident in the myenteric and submucosal plexuses [197]. In reindeer orally inoculated with CWD, PrP Sc accumulation is prominent in the myenteric and submucosal plexuses throughout the intestines [131]. In mule deer with CWD, reports vary and are limited. One report states that PrP Sc accumulation did not occur in the myenteric plexus following natural infection [121], while another report on PrP Sc in the enteric nervous system of mule deer states it is positive following natural infection [198].

Human Prion Diseases
There is evidence of expression of PrP C in the human enteric system [199]. However, there is no data to support PrP Sc accumulation in the enteric nervous system in people with CJD or GSS [200]. In a largescale survey of appendectomies in Britain, misfolded prion protein was found to be in the appendix of individuals, a handful of cases resulted in positive immunolabelling for PrP Sc in the appendix [201]. As the appendix in humans contains enteric neural tissue [202], future studies are necessary to investigate and characterize potential PrP Sc accumulation in the enteric nervous system of people with vCJD. PrP Sc accumulation is not investigated in cases of Kuru.

Conclusions
This review focused on three regions of the nervous system where neuroinvasion and subsequent PrP Sc accumulation is often different, in different strains of TSE within a given species. The retinal ganglion cells of the retina, the cerebellar cortex, and the enteric neurons of the foregut are three regions that may be useful to examine as additional TSE strains emerge and are needed to be characterized in the natural host.