Classical Scrapie, Atypical Scrapie and Evolutionary Biology

D. B. Adams
24 Noala Street, Aranda, ACT 2614, Australia
Tel: +61 2 6161 4825
E-mail: dadams@homemail.com

Abstract

Scrapie in sheep occurs in two forms. Classical scrapie, along with chronic wasting
disease in deer, differs from all other prion diseases in being contagious and occurring
as propagating epidemics. By contrast, atypical scrapie occurs sporadically and seems
to be non-contagious under natural conditions. This striking difference motivates the
present analysis with its emphasis on evolutionary biology complemented by inputs
from epidemiology, pathophysiology and the history of scrapie disease in Western
Europe from the 1700s onwards. Observations on strain behaviour and direct
demonstrations of natural selection establish that the scrapie agent and prions in
general are able to evolve. Foundations are thus set for proposing that atypical scrapie
can transform to classical scrapie under particular circumstances. Historical records,
which show a series of separate new beginnings of scrapie disease in Western Europe,
provide credible precedents and make the prospect of de novo scrapie epidemics an
issue for veterinary services. Such epidemics are likely to be extremely rare because
they require peculiar coincidences of factors related to sheep and their husbandry
environment. The functional failure that produces prion disease will be the crucial site
of impact and occurs at the level of protein quality control or proteostasis.
Accordingly, a preliminary approach to prevention and control can be based on
current knowledge of pathophysiology and epidemiology and built around a three-
stage model for the de novo genesis of a propagating epidemic of scrapie. The first
stage concerns the occurrence of atypical scrapie. The second stage concerns the
acquisition of communicability in prion populations provided by atypical scrapie
and the third stage concerns circumstances allowing disease transmission and the
initiation of a propagating epidemic. At present, almost anything that damages the
health and wellbeing of sheep is in contention as a component cause of de novo
classical scrapie.

Keywords

Scrapie, sheep, evolution, prion, proteostasis

1
Introduction

Scrapie disease in domesticated sheep and goats and chronic wasting disease (CWD)
in several species of wild-ranging deer possess the property of contagiousness or
communicability and occur as propagating epidemics (Fast and Groschup, 2013;
Williams, 2005). These features set them apart from other transmissible spongiform
encephalopathies (TSEs) or prion diseases such as Creutzfeldt-Jakob disease (CJD) in
humans, bovine spongiform encephalopathy (BSE), and atypical scrapie in sheep
(Hörnlimann et al., 2006), which are transmissible only by artificial means and in
imposed circumstances. Two strategically important questions arise from this crucial
difference. Firstly, how might a TSE transform from being transmissible only in
contrived conditions to being patently contagious and possessing inbuilt mechanisms
for exit and entry of the causative agent? Secondly, is atypical scrapie a predecessor
to classical scrapie and prospective hazard that can convert to a contagious form
under particular circumstances? Pursuit of these questions is impelled by a report
from Portugal on two outbreaks of classical scrapie that were was related in time and
place to a background incidence of atypical scrapie (Orge et al., 2010) and a case in
Italy where the pathological features characteristic of classical scrapie and atypical
(Nor98) scrapie were displayed simultaneously in a particular sheep (Mazza et al.,
2010). Given that scrapie in sheep is the archetype of the prion proteopathies, the
present exploration has a view to implications for comparative medicine.

Pathophysiology and evolutionary biology provide reasoning tools for addressing

these two strategically important questions and evidence for consideration comes
from knowledge of the epidemiology, aetiology and natural history of prion diseases.
Accordingly, the present effort outlines the case for natural selection in the scrapie
agent and other prions, reflects on the historical record of scrapie and compares the
natural history and pathophysiology of classical scrapie and CWD with that of
atypical scrapie. The aim is to identify potential prerequisites for the emergence of
classical scrapie from atypical scrapie or some other prion dysfunction and then to
explore the environmental influences, risk factors or component causes (Rothman and
Greenland, 2005) that may impel evolutionary processes. The intention is to enlighten
biosecurity measures against scrapie in places without the disease and inform
responses to sporadic occurrences of atypical scrapie.

Natural selection and the scrapie agent

Darwinian natural selection can be described by three inferences derived from five
facts or postulates (Mayr, 2001) and summed up in three key points: (1) Natural
selection is differential success in reproduction. (2) Natural selection occurs through
an interaction between the environment and the variability inherent among individual
organisms making up a population. (3) The product of natural selection is the
adaptation of populations of organisms to their environment (Campbell and Reece,

2
2002).

If a fundamental prerequisite is satisfied, this three-point picture of natural selection

can apply to the scrapie agent and to prions in general. The scrapie agent, a prion or a
protein that acquires alternative conformations that become self-propagating (Prusiner,
2013), is not an organism in the usual sense. However, the scrapie agent may fit as an
as an object of selection (Lewontin, 1970; Mayr, 2001). Objects of selection variously
labeled as units of selection (Lewontin, 1970), replicators (Dawkins, 1978), or
selectons (Mayr, 1997) are natural entities that can evolve because they have three
necessary capacities of variation, reproduction, and heritability (Lewontin, 1970; Hull,
1980). These capacities confer evolvability or the capacity to generate heritable,
selectable phenotypic variation (Kirschner and Gerhart, 1998).

The case for prions being objects of selection requires an explanation of the three
functions that define objects of selection. First, heritability in the biological, as
opposed to the statistical sense, refers to the faithful transmission of characteristics
through lineages of organisms, cells or molecules. Accordingly, species, groups,
organisms, cells, genes and self-reproducing molecules such as prions can exhibit
heritability. Second, the terms reproduction and replication are connected in that
replication is fundamental to the biological processes of reproduction of organisms
and the regeneration and repair of tissues and cells. Bonner (2007) refers to levels of
reproduction starting with chemical replication and exemplified by the template
replication of DNA, where one strand acts as a mould for another. An analogous
process applies to prions, where normal cellular prion protein (PrPC) is converted to
the diseased form of the prion protein (PrPSc) by a mechanism involving binding and
templated misfolding (Colby and Prusiner, 2011; Jucker and Walker, 2013; Wenborn
et al., 2015). (3) ‘Population’ has the statistical meaning of ‘an aggregate of creatures,
things, cases and so on’ (Campbell and Swinscow, 2009) and thus can apply to prions.

The natural history of scrapie in its principal host, the sheep, demonstrates that
variation, replication and heritability function in the scrapie agent and make it an
object of natural selection. Furthermore, observations on other prion diseases such as
BSE (Konold et al., 2015) and CWD (Perrott et al., 2012) and on laboratory models of
prion diseases in mice (Angers et al., 2010), hamsters (Kimberlin and Walker, 1978;
Crowell et al., 2015) and bank voles (Watts et al., 2014) signal that prions in general
classify as objects of natural selection.

Strains of prions

According to a working definition adapted from that used for malaria (Hommel and
Gilles, 1998), a strain is ‘a population of parasites [or communicable disease agents
such as the scrapie agent] derived from a primary isolate and possessing defined
characteristics’. Prion strains, including those for scrapie, may be differentiated

3
according to seven criteria or defined characteristics (Groschup et al., 2006). These
defined characteristics are (1) disease symptomatology, (2) incubation period, (3)
range of susceptible host species, (4) distribution and nature of lesions in the central
nervous system, (5) resistance to different methods of inactivation, (6) resistance to
digestion by proteinase-K digestion and sites of enzymatic cleavage, and (7) nature of
glycosylation of the prion molecule (Collinge et al., 1996).

The history of scrapie strains and their seminal contribution to knowledge of prion
diseases commences with studies on a primary isolate or passage line of scrapie
obtained from sheep and known as SSBP/1 (sheep scrapie brain pool 1). SSBP/1 dates
to 1945 and from a designed study in Scotland (Wilson et al., 1950), which extended
the findings of Cuillé and Chelle (1936) that the scrapie agent can be transmitted from
scrapie-infected to uninfected sheep by parenteral inoculation with preparations of
brain and spinal cord. Ten serial transmissions by Wilson et al. (1950) demonstrated
that a pathogen with the functions of replication and heritability was a necessary
cause of scrapie.

Dickinson (1976) recounted the subsequent history of SSBP/1 and reported that no
noticeable changes in the clinical signs, incubation periods or in the relatively mild
brain lesions were observed in experimentally infected sheep over 24 serial
transmissions of SSBP/1. Experience since then confirms the heritable characteristics
of the SSBP/1 strain as (1) clinical signs of fast onset, a short phase of ataxia and little
pruritis, (2) a distinctive neuropathology with sparse vacuolation and differences in
the distribution of PrPSc immunostaining in the brain compared with natural
indigenous scrapie, and (3) differences in genetic resistance with sheep encoding at
least one VRQ allele in their prion protein gene (PRNP) being susceptible to SSBP/1
and sheep encoding VRQ/VRQ and VRQ/ARQ being susceptible to indigenous
natural scrapie (Foster et al., 2013). These attributes provided a reliable means for
distinguishing SSBP/1 scrapie from indigenous natural scrapie and establishing that
scrapie transmits prenatally and by embryo transfer (Foster et al., 2013). To clarify,
notations ARQ and VRQ (plus AHQ, ARR and ARH) describe amino acid
substitutions at codons 136, 154 and 171 of the ovine PRNP gene and haplotypes that
are concerned with genetic resistance to classical scrapie (Dawson et al., 1998; Hunter
and Bossers, 2007).

Other strains of the scrapie agent exhibit the functions of heritability and replication
according to serial passage in sheep. One is CH1641, which was isolated in 1970
(Foster et al., 1988). CH1641 differs from SSBP/1 and indigenous natural scrapie (1)
in not being transmissible to mice with different alleles of the gene controlling the
scrapie incubation period (Foster et al., 1988), (2) in having a type C as opposed to a
type A or B glycoform pattern in its PrPSc (Hope et al., 1999), and (3) not being
transmissible by the oral route (Arsac and Baron, 2014).

Another scrapie strain exhibiting the functions of heritability and replication is Nor98,

4
which was first identified in Norway in 1998 (Benestad et al., 2003) and which can be
considered a subgroup of atypical scrapie (Benestad and Bratberg, 2006). Nor98 has
particularities as to clinical picture, genetic susceptibility, pathology and the nature of
its PrPSc (Benestad and Bratberg, 2006) and, unlike SSBP/1 scrapie and indigenous
natural scrapie, is not transmissible to the C57Bl, VM and RIII strains of inbred mice
(Benestad et al., 2008). Nor98 tends to occur in older animals around six years of age,
clinical signs can be subtle and vague. Signs include behavioural changes, emaciation
and ataxia and circling but not pruritus or wool loss. Nor98 can be found in sheep
with PrP genotypes that confer resistance to classical scrapie. The neuropathology
seen in Nor98 differs from that seen in classical scrapie. In particular, the dorsal
motor nucleus of the vagal nerve is unaffected; a finding of interest given that
neuroinvasion by classical scrapie may involve the enteric nervous system (van
Keulen et al., 2008). The PrPSc of Nor 98 is more susceptible to digestion by
proteinase K and detection requires immunochemical methods with higher sensitivity
than those for classical scrapie. Western blot profiles of Nor98 isolates show an
additional band at around 11-12 kDa (Benestad and Bratberg, 2006).

Other scrapie strains come with sufficient information to demonstrate heritability and
replication. Strains No. 13-7 and X124 were isolated from scrapie-infected sheep in
the USA (Bulgin et al., 2006; Hamir et al., 2009) and are set apart by different genetic
susceptibilities and incubation periods in sheep and different patterns of PrP Sc
accumulation (Moore et al., 2016). Variation among these five strains of the scrapie
agent parallels longstanding knowledge about variation in the natural history of
classical scrapie (Parry, 1966 and 1983; Ulvund, 2006). Masujin et al. (2009) and
Yokoyama et al. (2010) showed that brain homogenates from scrapie infected sheep
contained more than one strain of scrapie and that prion strain selection occurs upon
both inter- and intraspecies transmission.

Observations on scrapie disease as it occurs in sheep assign the functions of variation,

replication and heritability to the scrapie agent. The scrapie agent thus qualifies as an
object of selection and amenable to evolutionary processes.

Natural selection and prions

Investigation of prion strains in laboratory animal models began when the SSBP/1
strain of scrapie was transmitted to goats by intracerebral injection and two disparate
clinical pictures, ‘drowsy’ and ‘scratching’, emerged (Pattison and Millson, 1961).
Twenty-five years later, Bruce and Dickinson (1987) could refer to the existence of 15
different strains of scrapie derived from a range of cases in sheep and goats and
serially passaged in mice. Strains were differentiated according to reproducible
incubation periods in mice of defined genotypes and there was evidence for mutation
in particular strains. These and more recent observations made with laboratory animal
models of scrapie (Thackray et al., 2007; Thackray et al., 2008; Thackray et al., 2011;

5
Thackray et al., 2012) corroborate that scrapie strains possess functions of variation,
replication and heritability and corroborate the scrapie agent as a unit or object of
selection. Bruce and Dickinson (1987) applied knowledge existing at that time and
argued that the scrapie agent must possess an independently replicating genome based
on nucleic acids (the virino hypothesis). As stated earlier, chemical replication is now
known to go beyond nucleic acid entities and extends to proteins by means the prion
model of a misfolded self-propagating protein or protein aggregate (Uptain and
Lindquist, 2002; Wickner, 2004; Halfmann and Lindquist, 2010)

Observations on prion diseases other than scrapie and on the behaviour of their
associated strains (Solforosi et al., 2013) point to mechanisms that render prions
objects of selection. Angers et al. (2010) identified two strains of the CWD agent
according to their incubation times and neuropathology in mice and used differences
between deer species to demonstrate that strain selection and mutation was influenced
by residue 226 of the prion protein gene. Li et al. (2010) and Mahal et al. (2010)
studied prion strains in cultures of murine cell lines and showed that treatment with
swainsonine (an inhibitor of glycoprotein processing) led to heritable changes in the
phenotypic properties of prions that could be selectively amplified. Prion populations
responded in accord with the processes of natural selection and variants replicating
most abundantly in particular circumstances eventually became dominant. Similar
observations of natural selection have been corroborated for prions in a variety of
situations (for example Crowell et al., 2015; Ghaemmaghani et al., 2011 and 2013;
Vulin et al., 2012). Collinge (2016) encapsulated prions as a ‘dynamic collection or
cloud of misfolded protein assemblies that are maintained under biological selection
in a host, and its constituent tissues’ and existing as distinct strains with an ability to
adapt and mutate.

The depiction of prions as objects of selection equipped for variation, replication and
heritability is well grounded and justifies the thesis that non-contagious atypical
scrapie is a prospective epidemiological threat that can provide progenitors for the
prions causing contagious classical scrapie. Appraisal of this thesis can be assisted by
(1) a comparison of the characteristics of atypical and classical scrapie, (2) an
exploration of possible cellular and molecular mechanisms underlying the genesis of
atypical scrapie and possibilities for transformation to a communicable version of the
disease, (3) a search of the historical record of scrapie disease in sheep for indications
of an independent appearance of scrapie at different times and in different places, and
(4) reports on epidemiological aspects of atypical/Nor98 scrapie.

Comparison of atypical and classical scrapie

Classical scrapie, as opposed to the entity known as atypical scrapie, has epidemic
potential. The Panel on Biological Hazards of the European Food Safety Authority
expresses a consensus view that atypical scrapie ‘does not present, epidemiologically,

6
like an infectious disease’ (EFSA Panel on Biological Hazards, 2014). An
understanding of the basis for this crucial difference may come from a comparison of
the characteristics of classical and atypical scrapie (including Nor98 scrapie) as
regards PRNP genotypes affected, age of onset of the disease, symptomatology,
disease duration, neuropathological features, tissue distribution of PrP Sc and
infectivity, resistance of PrPSc to protease digestion and glycoform pattern.

Table 1 shows that the characteristics listed above differ between classical and
atypical scrapie and that differences occur at each level in the hierarchy of biological
organisation from molecule to organism. Some characteristics overlap between
classical and atypical scrapie and others show clear demarcations. Overlaps may
signify differing frequencies of variants within population of prions involved in
classical and atypical scrapie and imply scope for natural selection. Clear
demarcations between sets of characteristics may signify where gains-of-function in
prion populations are required for atypical scrapie to become contagious.

Table 1. Characteristics of classical scrapie and atypical scrapie (including disease caused
by the Nor98 agent)(from Parry, 1983; Benestad and Bratberg, 2006; Hörnlimann et al.,
2006; Ulvund, 2006: Benestad et al., 2008; Ulvund, 2008; Wemheuer et al., 2011; Fast and
Groschup, 2013).

Classical Scrapie Atypical scrapie (including

Nor98 scrapie)
Ovine genotypes VRQ and ARQ haplotypes most Nor 98 can occur in ARR genotypes.
affected* susceptible. ARR most resistant. High incidence of atypical scrapie in
AHQ genotype and low incidence in
ARQ genotype.
Age of onset of Between 2 and 5 years of age with On average 6.5 years old.
natural disease average age 3.5 years
Duration of disease 2 weeks to 6 months to death, rarely 6 weeks to 8 months to death
longer
Symptomatology Behavioural change, tremour, pruritus Weight loss, behavioural change
and locomotor disorder. (nervousness, anxiety), tremour
infrequent, pruritus not observed, and
locomotor disorder (ataxia, circling).
Nature and tissue PrPSc and infectivity occurs as constant PrPSc not detected outside the central
distribution of PrPSc feature in peripheral lymphoid tissue. nervous system or in the peripheral
and infectivity lymphoid tissue. Infectivity detected
in lymphoid tissue, nerves, and
muscles by bioassay with transgenic
mice.
Nature and Lesions in grey matter include Vacuolated grey matter predominates
distribution of vacuolation, neuronal loss and in the molecular layer of the
lesions in the central occasional astrocytosis. Lesions cerebellar cortex and also occurs
nervous system predominate in brainstem and occur at more rostrally in the basal ganglia
the level of the obex. Distribution of and cerebral cortex, Brainstem
lesions in more rostral areas of the brain lesions are insubstantial and no
varies widely. lesions are observed at the obex.

7
 Classical Scrapie
Nature and PrPSc occurs intra- and perineuronally,
distribution of PrPSc in association with glia and elsewhere.
deposits in central PrPSc deposits are a constant feature in
nervous system caudal brainstem, mainly at the level of
the obex and the dorsal motor nucleus of
the vagus nerve (DMNV). Extent of
PrPSc deposits depends on progression
of disease and occurs throughout the
CNS at clinical endpoint.
Atypical scrapie (including
Nor98 scrapie)
No PrPSc in neurons. PrPSc deposits
mainly in the cerebellar cortex
(especially in the molecular layer),
cerebral cortex and basal ganglia, in
the spinal tract nucleus of the
trigeminal nerve, and within white
matter throughout the brain. Dorsal
motor nucleus of the vagus nerve
(DMNV) without PrPSc.

Immunoblot pattern Three bands indicating unglycosylated,
monoglycosylated and diglycosylated
PrPSc with molecular masses between 18
and 30 kDa.
Four bands: Three indicate
unglycosylated,
monoglycosylated and diglycosylated
PrPSc with molecular masses between
18 and 30 kDa. A fourth band occurs
below 15 kDa.

Transmissibility to Yes Yes

transgenic mice
*Refers to variations in amino acid substitutions at codons 136, 154 and 171 of the ovine PRNP gene
and haplotypes that are concerned with genetic resistance to classical scrapie.

Genotypes of the PRNP gene explored in relation to either classical scrapie or atypical
scrapie have fundamentally different implications for pathogenesis. In the case of
classical scrapie, the fifteen genotypes classified into five types within the National
Scrapie Plan for Great Britain and other control programs (Hunter and Bossers, 2007)
link to a gradient of resistance against infection resulting from environmental
exposure to the scrapie agent. By contrast, the genotypic associations for atypical
scrapie connect to a situation involving the de novo and endogenous generation of
disease. Atypical scrapie has been seen most frequently in sheep with PRNP
genotypes tending towards resistance against classical scrapie (Benestad et al., 2008).
High prevalences of atypical scrapie have been observed in sheep with the AHQ/AHQ
genotype (Arsac et al., 2007; McIntyre et al., 2008) and in sheep with particular
polymorphisms at codon 141 of the PRNP gene (Moum et al., 2005).

Characters of classical scrapie and atypical scrapie that differ in degree rather than
kind are age of onset of disease, duration of disease and symptomatology. Differences
in these characters can be explained according to the concept of strain diversity in
prions (Collinge, 2016) and attributed in part to the action of variants within the
particular population of prions operating in an individual host.

As to age of onset, classical scrapie has been observed in the field in sheep from 1 to
11 years of age (Parry, 1983) and occurs most frequently in sheep between 2 and 5
years of age and at an average age of 3.5 years (Parry, 1983; Ulvund, 2008). Age of
onset of classical scrapie varies from flock to flock. For example, McIntyre et al.

8
(2008) recorded means from 30 flocks that ranged from 2.0 to 5.7 years. For atypical
scrapie, Ulvund points to 84 cases from nine different countries that record ages and
the mean age was 6.5 years. Lühken et al. (2007) analysed reports of scrapie in sheep
in Germany between January 2002 and February 2006 and found that 2 out of 20
cases (10%) of classical scrapie and 35 out of 60 (58%) cases of atypical scrapie
occurred in sheep older than 5 years. Fedaievsky et al. (2008) analysed data obtained
between 2002 and 2006 from 20 European countries under the European Union’s
active surveillance program for TSE in sheep and found that 127 out of 256 or 24% of
cases of classical scrapie and 231 out of 329 or 70% cases of atypical scrapie occurred
in sheep older than 5 years. In short, atypical scrapie tends to occur in older sheep and
differs from classical scrapie in this respect. The occurrence in older sheep may
reflect an aetiology related to host and environmental factors not exogenous infection
as in classical scrapie.

As to symptomatology, a thumbnail description of scrapie states that ‘the disease is

recognised by the clinical signs, which are variable, but generally start insidiously
with behavioural abnormalities then progress to more obvious neurological signs,
including pruritus and incoordination. Affected animals have poor body condition’
(OIE, 2017a). Ulvund (2006 and 2008) listed 33 different clinical signs of scrapie
from four different scientific reports and clustered them under headings of ‘general
signs’, ‘changes in behavior’, ‘changes in sensitivity’, ‘changes in locomotion’ and
‘other signs’. Ataxia and head tremor were the initial most dominating signs in Ireland,
while altered mental status, pruritus and wool loss were the initial most dominant
signs among Norwegian and Spanish sheep. In Italian sheep, both ataxia and pruritus
were seen frequently. Parry (1966) used over 1,200 individual clinical records to
classify the clinical expression of scrapie into five typical syndromes of ‘classical’
natural scrapie and five atypical syndromes of scrapie-like illness in sheep.

Scrapie in sheep has a remarkably diverse symptomatology that varies with flock,
breed, and region and points to factors in the host (genotype), disease agent (scrapie
strain) and environment (unknown). On this point, the clinical signs of atypical
scrapie in Norway were ataxia, anxiety and loss of body condition. Overt pruritus has
not been reported but the scratch reflex has (Konold et al., 2007) and may represent
nascent pruritus. The clinical signs in atypical scrapie have a pattern that may signal
the frequency and properties of certain prion variants in a particular strain and imply
scope for selection. The same consideration applies to the duration of disease and its
termination in death.

Characteristics of classical scrapie and atypical scrapie that differ in kind as opposed
to degree are (1) the tissue distribution of PrPSc and infectivity, (2) the nature and
distribution of lesions and PrPSc deposits in the central nervous system, and (3) the
immunoblot pattern. These differences align with capabilities present in contagious
classical scrapie but absent from atypical scrapie. Contagion in scrapie will be
precluded if prions are restricted to the central nervous system and have no access to

9
the portals of exit known to be involved in scrapie transmission. Once again,
differences between atypical and classical scrapie can be attributed to diversity among
and within prions strains. Furthermore, the accompanying ability of prion strains to
adapt and mutate (Collinge, 2016) provides a mechanism for gains and losses of
function, the acquisition of missing capabilities and the emergence of contagious
forms of scrapie.

Several studies have failed to detect PrPSc in peripheral lymphoid tissue in cases of
atypical/Nor98 scrapie. This finding is consistent with a spontaneous origin of PrPSc
in the brain but could also indicate that ingested PrPSc may bypass the lymphoid
system en route to the brain (reviewed in Benestad et al., 2008). The situation has
been clarified by highly sensitive transmission studies with transgenic mice
overexpressing the VRQ allele of the ovine PRNP gene. Such transmission studies
have detected infectivity in lymphoid tissues, nerves, and muscles in natural cases of
atypical/Nor 98 scrapie and also in atypical/Nor98 scrapie induced by intracerebral
inoculation (Andreoletti et al., 2011). The appearance of infectivity in peripheral
tissues following the induction of disease by intracerebral inoculation indicates that
infectivity outside the central nervous system originates from infectivity inside the
central nervous system. In natural cases of atypical scrapie, it is conceivable that
prions may form de novo both inside and outside the central nervous system.
Intracerebral injection causes extensive local hemorrhage and tissue necrosis and
leakage of inoculum into the ventricles and meninges (Hamir et al., 2002). The tissue
reactions involved would escalate the normal drainage of interstitial fluid to
peripheral tissue via the vascular, glial, and lymphatic immune pathways described
for the central nervous system (Weller et al., 2009; Aspelund et al., 2015; Engelhardt
et al., 2016). This drainage, as it occurs normally, can be proposed as a route of exit of
prions from the central nervous system in naturally occurring atypical scrapie.

Regardless of possible origin, scrapie prions that occur outside the central nervous
system in cases of atypical scrapie constitute a population in which mutation and
selection may lead to new and contagious strains of the scrapie agent. A crucial gain-
of-function would be increased affinity by the scrapie agent for the lymphoreticular
system. The consequential migratory and circulatory activity of lymphocytes and
mononuclear phagocytes would distribute infectivity throughout the body and transfer
it to and from prenatal and post-natal portals of exit and entry.

Predilections for particular portals of exit may be another important gain-of-function

in the possible evolution of contagious strains of scrapie. In this connection, portals of
exit demonstrated for the scrapie agent in sheep include excretions and ejecta such as
faeces (Everest et al., 2011: Terry et al., 2011), urine (Ligios et al., 2007; Rubenstein
et al., 2011), and foetal membranes (Pattison et al., 1972 and 1974; Onodera et al.,
1993; Race et al., 1998; Tuo et al., 2001 and 2002; Andreoletti et al., 2002; Alverson
et al. (2006); Lacroux et al., 2007); and secretions such as milk (Konold et al., 2008
and 2013; Lacroux et al., 2008; Maddison et al., 2009; Ligios et al., 2011), oral

10
secretions (Vascellari et al., 2007; Maddison et al., 2010; Gough et al. 2012) and
semen (Rubenstein et al., 2012). Tropisms for transmission routes that operate via
reproductive processes in sheep the may be pre-eminent in the acquisition of
communicability by prion populations in atypical scrapie. Reproductive processes
provide exceptional exposure to infection that is determined anatomically and
physiologically.

Cellular and molecular mechanisms involved in the genesis of

atypical scrapie

The functional failure at the root of all forms of scrapie and other prion disease relates
to a structurally abnormal form of the cellular prion protein (PrPC) that harms the
function of cells, tissues, organs and the body as a whole. Abnormal prions (PrPSc)
and their toxic intermediates (Sandberg et al., 2014) originate either exogenously as in
classical scrapie or endogenously as in atypical scrapie and proliferate by means of
seeded protein polymerization in which a PrPSc seed binds with PrPC and acts as a
template for misfolding (Colby and Prusiner, 2011). The resulting polymers split to
produce more seeds (Wenborn et al., 2015; Morales et al., 2016). Scrapie disease
reflects defective protein homeostasis and comes into existence when rates of PrP Sc
proliferation exceed rates of PrPSc clearance and there is an accumulation of PrPSc and
toxic intermediates.

Figure 1 sets out a general model for the cellular life cycle of the scrapie agent in
atypical and classical scrapie and shows factors involved in the proliferation and
clearance of PrPSc. As for all proteins, PrPC has a half-life and mechanisms for
protein homeostasis that operate through the proteostasis network (Balchin et al.,
2016; Jackson and Hewitt, 2016) produce a dynamic equilibrium between synthesis
and degradation that harmonises with changing demands for the prion protein.

11
Figure 1: Pathways for the production and removal of functional PrP C and pathways for the
misfolding and subsequent turnover of dysfunctional PrP Sc. Biosynthesis of PrPC occurs in
the ribosome and subsequent folding is assisted by molecular chaperone proteins. Points
where impairments to the proteostasis network may influence the kinetics of PrP Sc
accumulation are indicated.

Various factors related to protein biosynthesis and protein clearance by proteasomes

and lysosomes may influence the kinetics of PrPSc accumulation as follows: (1)
Protein folding is intrinsically error-prone (Balchin et al., 2016) and an induced
demand for PrPC may produce an amount of PrPSc that exceeds the adaptive capacity
of proteasomes and lysosomes. (2) An induced increase in the rate of PrPC misfolding
may exceed the adaptive capacity of clearance mechanisms in proteasomes and
lysosomes. (3) An induced decrease in the capacity of clearance mechanisms in
proteasomes and lysosomes may lead to a build-up of PrPSc produced at an otherwise
ordinary rate. (4) An induced increase in the production of PrPSc may coincide with an
induced decrease in the capacity of clearance mechanisms in proteasomes and
lysosomes. (5) The situation may be chaotic with unregulated fluctuations in the

12
production and misfolding of PrPC and the clearance of PrPSc.

The causal factors responsible for impaired proteostasis and the cellular abnormalities
that lead to the emergence of atypical scrapie may be genetic or acquired, or a
combination of the two. Kroemer et al. (2010) record that the pathways involved in
the lysosomal degradation of cytoplasmic organelles or cytosolic components are
affected by fluctuations in a broad range of external conditions. These include
physical factors (such as temperature and ultraviolet light), chemical factors (such as
ion concentrations, pH, oxygen tension, redox potentials and metabolite
concentrations), extracellular signals (such as contact-dependent signals, hormones,
cytokines and neurotransmitters) and microbial pathogens. Kroemer et al. (2010)
propose that when these factors exceed a certain threshold they can be considered
‘stresses’ that determine the function and survival of cells.

If the possibilities for impaired proteostasis outlined by Kroemer et al. (2010) are
extended to the genesis of atypical scrapie, virtually nothing in the genotype or
environment of sheep can be excluded as a causal factor. Best protection against the
emergence of atypical scrapie may occur through management to optimise the health
and wellbeing of sheep and thus to optimise their capacity to cope with stressors and
recover from the impacts of stress.

Indolizidine alkaloids are worthy of mention as one of many possible hazards for the
emergence of atypical scrapie. These alkaloids include swainsonine, which inhibits an
enzyme in lysosomes (α-D-mannosidase), leads to vacuolation in the nervous
system and produces a neurological syndrome in affected animals (Huxtable and
Dorling, 1982). Swainsonine is the poisonous principle in leguminous plants such
as Swainsona spp. in Australia, Astralagus spp. in North America and Oxytropis spp.
in China and can lead to considerable losses in grazing animals such as sheep
(Radostits et al., 2000). It is relevant that swainsonine has been used to demonstrate
the mutation and selection of prion strains in neuroblastoma cell culture and action
of evolutionary forces (Li et al., 2010). An analysis of the pathogenesis involved
in other plant-derived poisonings could point to other possibilities for the onset of
atypical scrapie.

Historical accounts of classical scrapie in sheep and their

implications for atypical scrapie

Parry (1983) stated that the clinical forms and epidemiological characteristics of
scrapie in sheep are such that “it is not mere chance that has ensured an extremely
high standard in the accounts of the disease published in many parts of north-western
Europe from 1750 onwards”. As a consequence, the histories of scrapie presented by
M’Gowan (1914), Parry (1983) and Schneider et al. (2008) can shed light on the
pattern of occurrence of scrapie according to time, place and population of sheep. In

13
doing so, these histories can assist in investigating the proposition that atypical
scrapie is prospective classical scrapie and in understanding the causative factors that
may allow the possibility of emergence.

The question put to the history of scrapie is whether the disease has appeared or
reappeared in different populations of sheep at different times and in different places.
Have outbreaks of scrapie in sheep populations always arisen through incursions of
the scrapie agent from disease reservoirs in other populations? In other words, to what
extent can recorded occurrences of scrapie be attributed to the migration of infected
animals or the movement of fomites or does the pattern of outbreaks signal a possible
de novo generation of the disease and a new reservoir of the scrapie agent? The
accounts of M’Gowan (1914), Parry (1983) and Schneider et al. (2008) collate
secondary and tertiary historical sources and do not build from first hand records. As
for all history, these accounts will not provide definitive answers to questions posed.
However, they give a crude impression of the past where the general tendencies of
scrapie can be gauged according to the epidemiological triangle of host, disease agent
and environment (Bhopal, 2002).

M’Gowan (1914) dwells upon the various names given to scrapie in Great Britain (e.g.
scratchie, rubbers, rickets, goggles, shakings, shrewcroft and cuddie trot) and refers to
the disease in south-western and south-eastern England, northernmost England and
southernmost Scotland. Scrapie, named as ‘rickets’, was reported in Lincolnshire
before 1750. Between 1750 and 1800, scrapie was reported in Cambridgeshire,
Huntingdonshire, Lincolnshire, Norfolkshire, Essex, Kent and Sussex in south-eastern
England, in Hampshire, Wiltshire, Dorsetshire and Somerset in south-western
England and in Lancashire in the northern sector of England. Scrapie was reported in
Devonshire and Berkshire between 1800 and 1850 and in Northumberland and
Roxburghshire between 1850 and 1900. The only breed mentioned by M’Gowan
(1914) is the Wiltshire and inbreeding is referred to as a causative factor for scrapie.

Parry (1983) extends the history of scrapie in Great Britain to the early 1980s, and
presents observations according to breed of sheep, derivation of the breed, time
periods of 50 years, geographical location and an arbitrary ordinal grade of scrapie
occurrences. Breeds provide a useful indicator of particular populations of sheep
husbanded and bred in relative isolation. Significantly, geographic distributions of
some scrapie-free breeds overlapped those of scrapie-affected breeds, implying
that agent-related and environment-related causative factors had minimal
influence. Nineteen breeds are recorded with scrapie and fifteen breeds are
recorded with no scrapie. In breeds such as the Norfolk Horn, Wiltshire Horn,
Dorset Horn, and Hampshire Down, scrapie appeared in reports between 1750 and
1800, disappeared for a century and reappeared after 1900. In breeds such as the
Welsh Mountain, Herdwick, Teeswater, Scottish Blackface, Shetland, Oxford
Down and Suffolk, scrapie was not recorded until after 1900. The pattern of

14
scrapie occurrence according to time, breed and place was unlike the pattern
displayed by other diseases of sheep and suggests a major influence from host-
related causative factors. The Spanish merino may have been important in
outbreaks of scrapie in France and Germany but can be discounted as a cause of
the multiple and geographically separated occurrences of scrapie seen in Great
Britain before 1787 when the Spanish merino first entered that country.

M’Gowan (1914) cites eight references that describe occurrences of scrapie in sheep
in Germany and France. Scrapie is reported in Upper Saxony before 1800 and three
references speak to the existence of scrapie in France during the 1800s. Parry (1983)
tabulates information on the prevalence of scrapie in France, Germany and the
Danube Valley from 1700 to 1980. In France, scrapie is reported as severe in merinos
at Rambouillet between 1780 and 1820, moderate but widespread (and extending to
milk sheep) between 1820 and 1880, and as sporadic outbreaks in many areas
between 1880 and 1950. In Germany, scrapie is reported as being present perhaps
between 1700 and 1750, occurring in merinos in Saxony and Silesia between 1750
and 1780, occurring as epidemics in Brandenburg and Saxony between 1780 and
1820, being severe in Electoral merinos in the east but not the south between 1820
and 1880, and declining then becoming rare and very rare between 1880 and 1950.
Reference is made to an outbreak in 1973 of scrapie in merinos in eastern Germany.
As to the Danube Valley, scrapie is reported as severe in Electoral merinos between
between 1820 and 1880 then declining to none after 1930.

Outbreaks of scrapie occurred after 1950 in Hungary and Bulgaria and can be
attributed to the scrapie agent present in Suffolks imported from England. In like
manner, earlier outbreaks of scrapie in merinos, particularly Electoral merinos and
their descendants, in France and Germany, can be attributed to incursions of the
scrapie agent. However, May in Germany in 1868, however, does not accept this as
the whole explanation. May, as referenced by M’Gowan (1924), delved deeper and
connected the outbreaks of scrapie in question to a list of possible ‘immediate’ and
‘remote’ causes related to imprudent breeding practices and adverse conditions of
husbandry. Importantly, occurrences of classical scrapie in Great Britain in the early
eighteenth century occurred in the absence of merinos and point to separate de novo
or autochthonous origins of the disease. This observation provides grounds for
claiming that similar separate autochthonous events may explain some of the
outbreaks of classical scrapie in France and Germany during the eighteenth and
nineteenth centuries.

Schneider et al. (2008) outline the early literature on the transmissible spongiform
encephalopathies and explore viewpoints about the causes and modes of transmission
of scrapie. Possible causes contemplated during the 1800s and late 1700s included
factors in the physical environment (thunderstorms, other exceptional atmospheric
events, humidity), nutritional environment (excessive or inadequate feeding, sodium

15
or potassium deficiency), biotic environment (e.g. ‘worm’ infection) and husbandry
environment (breeding from sheep too young or too old, cross-breeding, in-breeding
and tail docking). The most common view, however, was that host-related factors
(heredity) were paramount in occurrences of classical scrapie.

In sum, the histories offered by Parry (1983), M’Gowan (1914) and Schneider et
al. (2008) indicate that classical scrapie in Great Britain and continental Europe
appeared at different times and places as discrete episodes that were generated by
factors related to sheep and their environment and not by incursions of scrapie
from outside sources. The pattern observed is consistent with a sporadic form of
scrapie transforming to a communicable form and then to a full-blown epidemic
under the influence of three sets of factors acting in sequence (shown later in
Figure 2). The first set of factors would affect the occurrence of sporadic scrapie.
The second set would affect the acquisition of communicability and create the
prospect of a propagating epidemic. The third set of factors would allow
transmission and initiate a propagating epidemic of scrapie.

Epidemiological studies on atypical scrapie -Nor98 in sheep

The incidence and distribution of atypical scrapie has been investigated in sheep
populations in Norway (Hopp et al., 2006), Germany (Lühken et al., 2007), Great
Britain (Green et al., 2007; McIntyre et al., 2008; Del Rio Vilas et al., 2010; Ortiz-
Pelaez et al., 2016), Wales (Del Rio Vilas et al., 2011), France (Fediaevsky et al.,
2009 and 2010a), and in multiple European countries (Fediaevsky et al., 2008 and
2010b). The first report by Hopp et al. (2006) concluded that scrapie Nor98 differed
from classical scrapie in its distribution pattern and had a low to null communicability
in the field. Subsequent reports have drawn similar conclusions with none giving any
hint that scrapie Nor98 is infectious. For example, analysis of the records of active
surveillance for transmissible spongiform encephalopathies in small ruminants in
eleven European countries between 2004 and 2007 (Fediaevsky et al., 2010b)
prompted a conclusion that ‘atypical scrapie is not contagious or has a very low
transmissibility under natural conditions compared with classical scrapie’. Several
studies highlight the sporadic nature of atypical scrapie Nor98 (Hopp et al., 2006;
Lühken et al., 2007; Benestad et al., 2008; McIntyre et al., 2008; Del Rio Vilas et al.,
2010) and its resemblance to sporadic Creutzfeldt–Jakob disease in humans.

Lühken et al. (2007) described differences in the geographic-distribution, age-

distribution and breed-distribution of atypical and classical scrapie. Atypical scrapie
occurred across the whole of Germany whereas classical scrapie was restricted to
southern Germany. Classical scrapie was not observed in sheep over seven years old
whereas 43% of cases of atypical scrapie were found in sheep over seven years old.
Classical scrapie was recorded in three breeds and in crossbreeds whereas atypical

16
scrapie was found in eleven of twelve breed categories. Peculiarities of genetics were
also observed with the ARQ/ARQ PRNP genotype being most susceptible to classical
scrapie and the AF141RQ and AHQ haplotype having the highest risk for atypical
scrapie.

Epidemiological investigations into possible determinants or component causes of

atypical scrapie are available for sheep populations in Norway (Hopp et al., 2006),
France (Fediaevsky et al., 2009) and Great Britain (Del Rio Vilas et al., 2010). In
Norway, Hopp et al. (2006) found an increased risk of atypical scrapie Nor98 in
flocks given vitamin and mineral feed supplements, which could suggest either a
causative role for certain vitamins or minerals or the effect of factors that called for
feed supplementation. In France, Fediaevsky et al. (2009) found that sheep dairy
farms had a higher risk for atypical scrapie, which may reflect particular exposures
such as metabolic disorders related to lactation. In contrast to the findings of Hopp et
al., (2006), the feeding of vitamin and mineral supplements was associated with a
lower risk of atypical scrapie, which may be explained by differing environmental
conditions and differing interactions between diet, mineral intake and local stressors.
Fediaevsky et al. (2009) also found that sheep on farms operating under guidelines for
organic production and sheep fed corn silage had lower risks for atypical scrapie,
which was attributed to ‘less harsh farming conditions’. In Great Britain, Del Rio
Vilas et al. (2010) found that the organic status of sheep farms had no significant
impact on the risk of atypical scrapie. Breed had a significant impact on the risk of
atypical scrapie with Welsh Mountain and Cheviot breeds strongly associated
presence of the disease and Charolais sheep associated with absence of the disease.
Del Rio Vilas et al. (2010) also found that the greater the number of visits by
veterinarians the smaller the odds of atypical scrapie. Some explanations were offered
but the simplest may be a connection between the use of veterinary services and
diligence regarding animal health and wellbeing.

Information about the multitude of possible determinants of atypical scrapie may be

rudimentary. However, all listed accounts of atypical scrapie according to occurrences
in time, place and population indicate that cases occur without transmission from
sheep to sheep or between flocks. Atypical scrapie can be characterised as a
‘spontaneous disease with a genetic determinant and possible influence of
environmental and metabolic factors’ (Fediaevsky et al., 2009). Given that prion
strains constitute “a dynamic collection or 'cloud' of misfolded protein assemblies that
are maintained under biological selection in a host and its constituent tissues”
(Collinge, 2016), instances of atypical scrapie may provide populations of prions
susceptible to evolutionary processes.

Discussion

The hypothesis that atypical scrapie has an intrinsic potential for acquiring the

17
function of communicability and can be the progenitor of classical contagious scrapie
is supported by concepts from evolutionary biology and pathophysiology and by
studies of the epidemiology, aetiology and natural history of prion diseases. Accounts
of strain behaviour and direct demonstration of natural selection in action show that
prions in general and the prions involved with scrapie possess the basic functions of
variation, reproduction, and heritability (Lewontin, 1970; Hull, 1980), which underpin
evolvability (Kirschner and Gerhart, 1998). The mechanism for these functions
resides within the misfolded protein assemblies that constitute populations of disease-
causing prions (Collinge, 2016) rather than within a genome based on RNA or DNA.
As a consequence, transformation of atypical scrapie to contagious classical scrapie
by means of evolutionary processes can be proposed as a conceivable event with
intelligible causes related to the epidemiological triangle of disease agent, host and
environment (Bhopal, 2002). Since scrapie is the historical archetype of the prion
diseases, implications of prion evolvability go beyond the health of sheep and extend
to all species affected by diseases that impair processes for protein quality control
(Ciechanover and Kwon, 2017; Hartl, 2017; Sontag et al., 2017) and which involve
prions or ‘proteinaceous nucleating particles’ (Walker and Jucker, 2015).

Historical accounts of scrapie (M’Gowan, 1914; Parry, 1983; Schneider et al.,

2008) indicate that propagating epidemics of the disease can have new autochthonous
beginnings at different times and places and in different populations of sheep. In
doing so they endorse the notion that atypical scrapie can give rise to classical scrapie.
Occurrences of scrapie in Great Britain before the introduction of stocks of merinos in
1787 are crucial. They show that the origins of scrapie need not be uniquely attributed
to the merino breed as suggested by experience with scrapie in eighteenth and
nineteenth century France and Germany where outbreaks were associated with
merinos (particularly Electoral merinos) and their descendants. May (1868)
suggested that these outbreaks were connected to some possible ‘immediate’ and
‘remote’ causes that arose from adverse conditions and practices in the new and
unfamiliar husbandry environment of merino sheep.

May’s thoughts in 1868 on the origin of classical scrapie in eighteenth and nineteenth
century France and Germany accord with the present hypothesis that propagating
epidemics of classical scrapie can arise from sources in atypical scrapie. Three stages
can be envisaged for the process of transformation from one manifestation of scrapie
to another (Figure 2). Stage one would concern the induction of atypical scrapie.
Stage two would concern the induction of classical scrapie from atypical scrapie and
stage three would concern the induction of a propagating epidemic. Knowledge from
epidemiology and pathology can illuminate the factors involved in these three stages
and inform the development of biosecurity measures and ways and means of
optimising flock health.

18
 Stage 3:
Stage 1: Induction of Stage 2: Progression Induction of a propagating
atypical scrapie to classical scrapie epidemic

Basis: Functional failure in Basis: Acquisition and Basis: Effectual

proteostasis and amplification of the transmission of
accumulation of misfolded function of the scrapie agent
scrapie prions causing communicability in
disease populations of prions
provided by atypical
scrapie

Putative causes Putative causes

Putative causes
Sheep-environment Circumstances that open
Increase in scrapie prions with affinity
interactions and imposts on opportunities for prenatal
for lymphoreticular cells allowing for
physiological stability transmission from sources in
distribution throughout body and to
(allostatic load) rams and ewes
portals of exit
Infectious or non-infectious Circumstances that open
Neuroinflammation increasing the
neurological disorders opportunities for post-natal
traffic of immune cells to and from the
central nervous system transmission by means of the
Exposure to mycotoxins, scrapie agent in faeces, urine,
phytotoxins and milk and oral secretions
Intercurrent infection (e.g. lentivirus)
allowing increased contact between
scrapie prions and lymphoreticular

Figure 2: The three-stage process required for the de novo generation of a propagating
epidemic of classical scrapie in sheep showing the conceptual basis for each stage and an
intimation of possible causes or risk factors.

An understanding of the determinants of atypical/Nor98 scrapie and the first stage of

a possible progression to a full-blown scrapie epidemic can come from epidemiology
and pathology. Epidemiological studies define atypical/Nor98 scrapie as a non-
communicable version of scrapie (Hopp et al., 2006: Lühken et al., 2007; Fediaevsky
et al., 2010b), which tends to occur in older animals and has a longer duration of
illness than classical scrapie. In brief, epidemiological studies indicate that the
foundations of atypical/Nor98 lie within sheep and their environment and interactions
between the two.

Proteostasis and the accumulation of misfolded protein that characterise prion

diseases such as scrapie are points of interest in the genesis of atypical scrapie and the
situation can be explored with help from the concepts of allostasis and allostatic load
(Goldstein and Kopin, 2007: McEwen and Wingfield, 2010; Goldstein, 2012;
McEwen et al., 2015). These concepts unify notions of stress, homeostasis and
physiological stability or eustasis, as opposed to cacostasis (Tsigios et al., 2016).
Allostasis refers to active processes that underlie adaptation to intrinsic and extrinsic
stressors and allostatic load refers to the cumulative effects of exposure to stressors
and their adverse consequences in pathophysiology and dysfunction (Tsigios et al.,
2016; National Academies of Sciences, Engineering, and Medicine. 2017). Allostatic

19
load evokes the notion of wear and tear and bears upon the second law of
thermodynamics and the need for continual repair and regeneration in living
organisms. Proteostasis or the quality controlled cellular processes for the production
and turnover of proteins (Hartl, 2017; Sontag et al., 2017) is central to repair and
regeneration and thus falls within the scope of allostasis.

The functional failure that leads to atypical scrapie may occur at three key places in
the process of proteostasis as depicted in Figure 1. Firstly, an induced demand for
PrPC may be accompanied by amounts of PrPSc that exceed the adaptive capacity of
protein clearance mechanisms. Secondly, higher amounts of PrP Sc resulting from
increases in misfolding may overwhelm the clearance mechanisms through
proteasomes and by autophagy. Thirdly, clearance mechanisms or other components
of the protein quality control system (Sontag et al., 2017) may malfunction or any or
all of the three possibilities may interact. Alternatively, there may be a general
collapse in proteostasis occasioned by allostatic load.

Reviews of the biological function of the cellular prion protein (PrP C) (Castle and Gill,
2017; Linden, 2017; Wulf et al., 2017) do not hint at circumstances that may increase
rates of production and heighten the risk of the misfolded conformer (PrPSc). A
generalised causal link between neuroinflammation and neurodegeneration marked by
misfolded protein (Ransohoff, 2016) implies that virtually any infectious or non-
infectious exposure that leads to neurological disease in sheep can be regarded as a
potential risk factor for atypical scrapie and the list is extensive (Finnie et al., 2011;
Kessell et al.,2011a; Kessell et al., 2011b). More specifically, certain environmental
exposures could increase rates of misfolding of PrPC and lead to atypical scrapie
given that swainsonine, an indolizidine alkaloid, was used to induce mutations in
prions and to demonstrate Darwinian evolution in prion strains (Li et al., 2010; Mahal
et al., 2010).

Swainsonine is a mycotoxin synthesized by endophytic fungi and associated with

poisonous plants such as Swainsona spp., Astralagus spp. and Oxytropis spp., which
cause neurological diseases in sheep and other ruminants (Radostits et al., 2000).
Sheep are universally exposed to mycotoxins, phytotoxins and environmental
toxicants and it is inconceivable that none would have consequences for the
misfolding of protein and similar implications for the onset of atypical scrapie.
Malfunctions in protein clearance mechanisms that may trigger atypical scrapie are
conceivable accompaniments to the inherited lysosomal disorders that occur in sheep
(Jolly and Walkley, 1997).

Possible impacts of allostatic load on proteostasis and a consequential increased risk

of atypical scrapie can be viewed from a comparative medicine or ‘one health’
perspective and with reference to the example of Alzheimer’s disease in humans. Like
scrapie, Alzheimer’s disease is a proteopathy and a prion disease hinging on the
seeded aggregation and impaired clearance of disease-specific proteins (Walker and

20
Jucker, 2015). The incidence of Alzheimer’s disease in white women in the USA
from 1986 to 2006 was positively associated with the social gradient; that is, women
lower in the social or wealth spectrum had higher incidences of the disease (Montez,
and Zajacova, 2013). The social gradient builds on the social determinants of health
or the circumstances in which people are born, grow up, live, work and age, and the
systems put in place to deal with illness (Wilkinson and Marmot, 2003). In doing so,
the social gradient reflects chronic stress or allostatic load. A similar impact of
chronic stress or allostatic load applies to scrapie in sheep where a lower frequency of
the disease was associated with ‘less harsh farming conditions’ (Fediaevsky et al.,
2009).

Stage two in the notional progression from atypical scrapie to classical scrapie and
then to a full-blown epidemic of scrapie concerns the acquisition of communicability
in populations of prions generated in atypical scrapie. Investigations of atypical
scrapie have detected no animal-to-animal transmission of the disease and lack of
communicbility can be deemed as the defining feature of atypical scrapie. The
distinction between communicability and transmissibility has fundamental importance.
Contagiousness goes beyond transmissibility and entails inbuilt mechanisms for exit
of the causative agent from one animal and entry to another. Simple transmissibility
does not require mechanisms for portals of exit and entry of the disease agent and is
demonstrated by experimental inoculation and by instances where artificial exposures
caused common-source as opposed to propagating epidemics. Examples are bovine
spongiform encephalopathy (BSE), where the large-scale transboundary epidemic was
driven by feedstuffs containing products derived from infected cattle, and iatrogenic
Creutzfeldt-Jakob disease (iCJD) where outbreaks resulted from contaminated
surgical instruments, contaminated extracts of human pituitary glands and
contaminated implants of corneas and dura mater (Hörnlimann et al., 2006). If the
circumstances of BSE and iCJD were to recur with atypical scrapie in sheep, any
ensuing common-source outbreak would expand populations of scrapie prions
available for evolutionary selection and favour the possible emergence of
communicability and classical scrapie.

Atypical and classical scrapie have differences in the tissue distribution of misfolded
prions (PrPSc) and infectivity that may align with contgiousness. PrPSc and infectivity
are present as a constant feature in the lymphoreticular tissues and cells of sheep with
classical scrapie. In contrast, PrPSc is restricted to the central nervous system and not
detected in the lymphoreticular tissues and cells of sheep with atypical scrapie. This
difference is cardinal because the migratory behaviour of lymphocytes and
mononuclear phagocytes (Parham, 2015) provides a means for disseminating the
scrapie agent throughout the body and to the multiple portals of exit that operate
either prenatally or post-natally. Exosomes may also be involved in the bodily
distribution of the scrapie agent (Fevrier et al., 2004). Significantly, transmission
studies with transgenic mice have detected infectivity in lymphoid tissues, nerves and
muscle of sheep with atypical scrapie occurring naturally or induced by intracerebral

21
inoculation (Andreoletti at al., 2011). Accordingly, atypical scrapie is associated with
a small population of prion variants with an affinity for lymphoreticular cells and a
propensity for communicability that could be expanded by evolutionary processes.

Current knowledge allows a broad appreciation of where and when scrapie prions
could move from the central nervous system, what influences the presence of prions
in lymphoreticular cells and how atypical scrapie could transform to classical scrapie.
The scrapie agent could move from the central nervous system through cerebrospinal
fluid (CSF) or interstitial fluid and in exosomes or cells, particularly immune cells,
which are known to traffic through nervous tissue as part of immune surveillance
(Wraith and Nicholson, 2012; Ransohoff and Engelhardt, 2012; Engelhardt et al.,
2016). CSF drains from the central nervous system through a network of lymphatic
vessels in the dura mater and then to cervical lymph nodes (Aspelund et al., 2015;
Louveau et al., 2015) and interstitial fluid drains to lymph nodes by means of
pathways within the walls of cerebral capillaries and arteries (Engelhardt et al., 2016;
Kipnis, 2016). Exchange between interstitial fluid and CSF occurs through the
glymphatic system, which comprises connected periarterial and perivenous spaces
(Engelhardt et al., 2016; Kipnis, 2016). Neuroinflammation may result in increased
trafficking of immune cells through the central nervous system (John et al., 2014) and
an increased opportunity for prions associated with atypical scrapie to spread from the
brain. Thus, virtually all infectious or non-infectious exposures that lead to
neurological disease in sheep (Finnie et al., 2011; Finnie et al.,2011a; Kessell et al.,
2011) can be reiterated as potential risk factors for the transformation of atypical to
classical scrapie.

Another set of potential risk factors for the transformation of atypical to classical
scrapie relates to increased affinity for lymphoreticular cells in prion populations.
Once again the field of candidate risk factors is large because the possibilities include
conditions accompanied by inflammation or perturbations to the lymphoreticular
system, or both. Precedents come from observations in sheep co-affected with scrapie
and chronic inflammatory conditions (Maestrale et al., 2013) and sheep with lentiviral
infections (Lacroux et al., 2008; Salazar et al., 2010; Ligios et al., 2011).

The third stage in a possible transformation of atypical scrapie to classical scrapie is a

propagating epidemic driven by scrapie prions with the function of communicability
(see Figure 2). Risk factors or component causes for this stage spring from
transmission, which is known to occur both prenatally and post-natally. Prenatal
transmission is firmly established and can result from the scrapie agent in semen or in
the dam, and the conceptus can be infected at any time from oogenesis onwards
(Foster et al., 2013; Garza et al., 2011; Rubenstein et al., 2012; Spiropoulos et al.,
2014; Adams, 2016). Post-natal transmission can occur through environmental
exposure to the scrapie agent derived from ovine excretions and ejecta such as faeces
(Terry et al., 2011; Everest et al., 2011), urine (Rubenstein et al., 2011; Ligios et al.,
2007), and foetal membranes (Alverson et al., 2006; Andreoletti et al., 2002; Lacroux

22
et al. (2007), Onodera et al. (1993), Pattison et al. (1972 and 1974a), Race et al.
(1998),Tuo et al. (2001 and 2002). ) and in ovine secretions such as milk ( Konold et
al (2008), Lacroux et al (2008), Maddison et al. (2009), Ligios et al (2011), Konold et
al. (2013a), oral secretions (: Gough et al. 2012; Maddison et al., 2010), Vascellari et
al. (2007), and semen (Rubenstein et al., 2012).

Infectivity (the proportion of exposures resulting in infection) was analysed in

historical records from six scrapie infected flocks (2,060 offspring and 606 cases of
scrapie) to gauge the relative impact of prenatal and post-natal transmission pathways
(Adams, 2016). Transmission from sire to conceptus recorded an infectivity of 43%
and transmission from dam to conceptus plus transmission from dam to neonate had
an infectivity of 44%. These prenatal infectivities outweigh the 12% infectivity
associated with post-natal transmission of scrapie agent from the environment. They
signal that pre-emptive attention to risk factors associated with breeding can block the
possibility of a propagating epidemic arising from a rare occurrence of atypical
scrapie. Broad preventive measures can build from earlier recommendations
(Detwiler and Baylis, 2013) and emphasise breeding only with sheep in robust health
and the avoidance of lambing in confined spaces where contact with contaminated
ejecta and excretions may be unavoidable.

Given that the scrapie agent and prions in general are objects of selection and can
evolve, de novo propagating epidemics of scrapie disease are conceivable when
particular conditions and risk factors coincide in the three-stage process for
transformation depicted in Figure 2. As a result, probabilities will be multiplicative,
implying that the overall probability of a de novo scrapie epidemic will be minuscule.
However, the possibility of de novo epidemics constitutes a clear-cut concern for
veterinary services everywhere and by reason of the ecological fallacy or fallacy of
division, This informal fallacy refers to the 'erroneous transference of an attribute
from a whole (or a class) onto its parts (or members)' (Hurley, 2003) and applies to
epidemiology, where errors will arise when the aggregated behaviour of global
populations is extrapolated to sub-populations without regard to circumstances
(Bhopal, 2002; Bonita et al., 2016). The model for veterinary services promulgated by
the World Organisation for Animal Health (OIE, 2017) proposes ongoing capacity-
building and follows the precepts of comparative medicine. These precepts
encompass evolutionary biology (LeGrand and Brown, 2002; Nesse et al., 2010) and
can thus create a capacity for clarifying and managing sub-population susceptibilities
to atypical scrapie and their possible consequences..

The component causes of atypical scrapie and its possible progression to classical
scrapie are very broad and include virtually anything that impinges on the
physiological stability or allostasis of sheep and which may harm proteostasis or
favour the selection of prion variants with the property of communicability. For this
reason, actions that prevent atypical scrapie will fortify efforts to enhance the health,
welfare and productivity of sheep and will assist towards the thermodynamic

23
efficiency required for sustainable regenerative agriculture (Gliessman, 2007).

References

Adams, D. B. 2016. Prenatal transmission of scrapie in sheep and goats: A case study
for veterinary public health. Open Vet. J. 6, 194-214.

Alverson, J., O’Rourke, K.I. and Baszler, T.V. 2006. PrPSc accumulation in fetal
cotyledons of scrapie-resistant lambs is influenced by fetus location in the uterus.
J. Gen. Virol. 87, 1035–1041.

Andreoletti, O., L. Orge, S.L. Benestad, V. Beringue, C. Litaise, S. Simon, A. Le Dur,

H. Laude, H. Simmons, S. Lugan, F. Corbiere, P. Costes, N. Morel, F. Schelcher,
C. Lacroux. 2011. Atypical/Nor98 scrapie infectivity in sheep peripheral tissues.
PLoS Pathogens. 7, e1001285.

Andreoletti, O., Lacroux, C., Chabert, A., Monnereau, L., Tabouret, G., Lantier, F.,
Berthon, P., Eychenne, F., Lafond-Benestad, S., Elsen, J.M. and 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, 2607–2616.

Angers, R.C., Kang, H.E., Napier, D., Browning, S., Seward, T., Mathiason, C.,
Balachandran, A., McKenzie, D., Castilla, J., Soto, C., Jewell, J., Graham, C.,
Hoover, E.A. and Telling, G.C. 2010. Prion strain mutation determined by prion
protein conformational compatibility and primary structure. Science. 328, 1154–
1158.

Arsac, J.N. and T. Baron. 2014. Distinct transmissibility features of TSE sources
derived from ruminant prion diseases by the oral route in a transgenic mouse
model (TgOvPrP4) overexpressing the ovine prion protein. PLoS One. 9, e96215.

Arsac, J.N., O. Andreoletti, J.M. Bilheude, C. Lacroux, S.L. Benestad, T. and Baron.
2007. Similar biochemical signatures and prion protein genotypes in atypical
scrapie and Nor98 cases, France and Norway. Emerg. Infect. Dis. 13, 58-65.

Aspelund, A., Antila, S., Proulx, S.T., Tine, V.K., Karaman, S., Detmar, M., Wiig, H.
and Alitalo, K. 2015. A dural lymphatic vascular system that drains brain
interstitial fluid and macromolecules. J. Exp. Med. 212, 991-999.

Balchin, D., Hayer-Hartl, M. and Hartl, F.U. 2016. In vivo aspects of protein folding
and quality control. Science. 353, aac4354.

24
Benestad, S.L. and Bratberg, B. 2006. Atypical scrapie-Nor98. In Prions in Humans
and Animals, Eds. Hörnlimann, B., Riesner, D. and Kretschmar, H. Berlin: De
Gruyter, pp: 630-634.

Benestad, S.L., Arsac, J.N., Goldmann, W. and Noremark, M. 2008. Atypical/Nor98

scrapie: properties of the agent, genetics, and epidemiology. Vet. Res. 39, 19.

Benestad, S.L., Sarradin, P., Thu, B., Schonheit, J., Tranulis, M.A., and Bratberg, B.
2003. Cases of scrapie with unusual features in Norway and designation of a new
type, Nor98. Vet. Rec. 153, 202-208.

Bhopal, R.S. 2002. Concepts of Epidemiology: An integrated introduction to the ideas,

theories, principles and methods of epidemiology. Oxford: Oxford University
Press.
Bonita, R., Beaglehole, R. and Kjellström, T. 2016. Basic Epidemiology 2nd Ed.
Geneva: World Health Organization.

Bonner, J.T. 2007. Reproduction. Encyclopædia Britannica.15th Ed. Chicago:

Encyclopædia Britannica Inc.

Bruce, M.E. and Dickinson, A.G. 1987. Biological evidence that scrapie agent has an
independent genome. J. Gen. Virol. 68, 79-89.

Bulgin, M.S., Sorensen ,S.J. and Matlock, M.E. 2006. Association between incubation
time and genotype in sheep experimentally inoculated with scrapie-positive brain
homogenate. Am. J. Vet. Res. 67, 498-504.

Campbell, M.J. and Swinscow, T.D.V. 2009. Statistics at Square One. Oxford: BMJ
Publishing Group Limited and John Wiley and Sons Limited.

Campbell, N.A. and Reece, J.B. 2002. Biology, 6th Ed. San Francisco: Benjamin
Cummings.

Castle, A.R. and Gill, A.C. 2017. Physiological Functions of the Cellular Prion
Protein. Front. Mol. Biosci. 4, 19.

Ciechanover, A. and Kwon, Y.T . 2017. Protein Quality Control by Molecular

Chaperones in Neurodegeneration. Front. Neurosci. 11, 185.

Colby, D.W. and Prusiner, S.B. 2011. Prions. Cold Spring Harb. Perspect. Biol. 3,
a006833.

Collinge, J. 2016). Mammalian prions and their wider relevance in neurodegenerative

diseases. Nature 539, 217-226.

25
Collinge, J., Sidle, K.C., Meads, J., Ironside, J. and Hill, A.F. 1996. Molecular
analysis of prion strain variation and the aetiology of ‘new variant’ CJD. Nature
383, 685-690.

Crowell, J., Hughson, A., Caughey, B. and Bessen, R.A. 2015. Host determinants of
prion strain diversity independent of prion protein genotype. J. Virol. 89, 10427-
10441.

Cuille, J. and Chelle, P.L. 1938. La tremblante du mouton est bien inoculable. C. R.
Acad. Sci. (Paris) 206, 78–79.

Dawkins, R. 1978. Replicator selection and the extended phenotype. Z Tierpsychol.

47, 61–76.

Dawson, M., Hoinville, L.J., Hosie, B.D. and Hunter, N. 1998. Guidance on the use of
PrP genotyping as an aid to the control of clinical scrapie. Scrapie Information
Group. Vet. Rec. 142, 623-625.

Del Rio Vilas VJ, Vink WD, Hubbard R (2010) A case-control study of atypical
scrapie in GB sheep flocks. Prev. Vet. Med. 96, 241-251.

Del Rio Vilas, V.J., Ancelet, S., Abellan, J.J., Birch, C.P. and Richardson, S. 2011. A
Bayesian hierarchical analysis to compare classical and atypical scrapie
surveillance data; Wales 2002-2006. Prev. Vet. Med. 98, 29-38.

Detwiler, L. A. and Baylis, M. 2003. The epidemiology of scrapie. Rev. Sci. Tech. 22,
121-143.

Dickinson, A.G. 1976. Scrapie in sheep and goats. In Slow Virus Disease of Animals
and Man. Ed.,.Kimberlin, R.H. Amsterdam: North-Holland Publishing Company.
pp: 209-41.

EFSA Panel on Biological Hazards. 2014. Scientific opinion on the scrapie situation
in the EU after 10 years of monitoring and control in sheep and goats. EFSA
Journal 12, 3781.

Engelhardt, B., Carare, R.O., Bechmann, I., Flugel, A., Laman, J.D. and Weller, R.O.
2016. Vascular, glial, and lymphatic immune gateways of the central nervous
system. Acta. Neuropathol. 132, 317-338.

Everest, S. J., Ramsay, A. M., Chaplin, M. J., Everitt, S., Stack, M. J., Neale, M. H.,
Jeffrey, M. S., Moore, J. S., Bellworthy, J. and Terry, L. A. 2011. Detection and

26
 localisation of PrP in the liver of sheep infected with scrapie and bovine
spongiform encephalopathy. PLoS One 6, e19737.

Fast, C. and Groschup, M.H. 2013. Classical and atypical scrapie in sheep and goats.
In Prions and Diseases, Volume 2, Animals, Humans and the Environment. Eds.
Zou, W-Q. and Gambetti. P. New York: Springer, pp: 15-44.
Fediaevsky, A., Gasqui, P., Calavas, D. and Ducrot, C. 2010a. Discrepant
epidemiological patterns between classical and atypical scrapie in sheep flocks
under French TSE control measures. Vet. J. 185, 338-340.

Fediaevsky, A., Maurella, C., Noremark, M., Ingravalle, F., Thorgeirsdottir, S., Orge,
L.,Poizat, R., Hautaneimi, M., Liam, B., Calavas, D., Ru, G. and Hopp B. 2010b.
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., Morignat, E., Ducrot, C., and Calavas, D. 2009. A case-control study
on the origin of atypical scrapie in sheep, France. Emerg. Infect. Dis. 15, 710-718.

Fediaevsky, A., Tongue, S.C., Noremark, M., Calavas, D., Ru, G. and 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.

Fevrier, B., Vilette, D., Archer, F., Loew, D., Faigle, W., Vidal, M., Laude, H. and
Raposo, G. 2004. Cells release prions in association with exosomes. Proc. Natl.
Acad. Sci. U S A 101, 9683-9688.

Finnie, J.W., Windsor, P.A. and Kessell, A.E. 2011. Neurological diseases of
ruminant livestock in Australia. I: General neurological examination, necropsy
procedures and neurological manifestations of systemic disease, trauma and
neoplasia. Aust. Vet. J. 89, 243-246.

Finnie, J.W., Windsor, P.A. and Kessell, A.E. 2011. Neurological diseases of
ruminant livestock in Australia. II: toxic disorders and nutritional deficiencies avj_793

Aust. Vet. J. 89, 247-252.

Foster, J.D. and Dickinson, A.G. 1988. The unusual properties of CH1641, a sheep-
passaged isolate of scrapie. Vet. Rec. 123, 5-8.

Foster, J.D., Goldmann, W. and Hunter, N. 2013. Evidence in sheep for pre-natal
transmission of scrapie to lambs from infected mothers. PLoS One 8, e79433.

27
Garza, M.C., Fernandez-Borges, N., Bolea, R., Badiola, J.J., Castilla, J. and Monleon,
E. 2011. Detection of PrPres in genetically susceptible fetuses from sheep with
natural scrapie. PLoS One 6, e27525.

Ghaemmaghami, S., Colby, D.W., Nguyen, H.O., Hayashi, S., Oehler, A., DeArmond,
S.J. and Prusiner, S.B. 2013. Convergent replication of mouse synthetic prion
strains. Am. J. Pathol. 182, 866-874.

Ghaemmaghami, S., Watts, J.C., Nguyen, H.O., Hayashi, S., DeArmond, S.J. and
Prusiner, S.B. 2011. Conformational transformation and selection of synthetic
prion strains. J. Mol. Biol. 413, 527-542.

Gliessman, S.R. 2007. Agroecology: the ecology of sustainable food systems. Boca
Raton, Florida: CRC Press.

Goldstein, D.S. 2012. Stress, allostatic load, catecholamines, and other

neurotransmitters in neurodegenerative diseases. Cell. Mol. Neurobiol. 35, 661-
666.

Goldstein, D.S. and Kopin , I.J. 2007. Evolution of concepts of stress. Stress 10, 109-
120.

Gough, K.C., Baker, C.A., Rees, H.C., Terry, L.A., Spiropoulos, J., Thorne, L. and
Maddison, B.C. 2012. The oral secretion of infectious scrapie prions occurs in
preclinical sheep with a range of PRNP genotypes. J. Virol. 86, 566-571.

Green, D.M., Victor, J., Birch, C.P., Johnson, J., Kiss, I.Z., McCarthy, N.D. and Kao,
R.R., 2007. Demographic risk factors for classical and atypical scrapie in Great
Britain. J. Gen. Virol. 88, 3486-3492.

Groschup M.H., Gretzschel, A. and Kuczius, T. 2006. Prion strains In Prions in

Humans and Animals, Eds. Hörnlimann, B., Riesner, D. and Kretschmar, H.
Berlin: De Gruyter, pp: 166-186.

Halfmann, R. and Lindquist, S. 2010. Epigenetics in the extreme: prions and the
inheritance of environmentally acquired traits. Science 330, 629-632.

Hamir, A.N., Kunkle, R.A., Richt, J.A., Greenlee, J.J. and Miller, J.M. 2009. Serial
passage of sheep scrapie inoculum in Suffolk sheep. Vet. Pathol. 46, 39-44.

Hamir, A.N., Miller, J.M., Stack, M.J. and Chaplin, M.J. 2002. Failure to detect
abnormal prion protein and scrapie-associated fibrils 6 wk after intracerebral
inoculation of genetically susceptible sheep with scrapie agent. Can. J. Vet. Res.
66, 289-294.

28
Hartl, F.U. 2017. Protein misfolding diseases. Annu. Rev. Biochem. 86, 21-26.
Hommel, M. and Gilles, H.M. 1998 Malaria. In Topley and Wilson’s Microbiology
and Microbial Infections, Volume 5 Medical Parasitology. Eds Cox, F.E.G.,
Kreier, J.P. and Wakelin, D. London: Hodder Arnold. pp: 361-410.

Hope, J., Wood, S.C., Birkett, C.R., Chong, A., Bruce, M.E., Cairns, D., Goldmann,
W., Hunter, N. and Bostock, C.J. 1999. Molecular analysis of ovine prion protein
identifies similarities between BSE and an experimental isolate of natural scrapie,
CH1641. J. Gen. Virol. 80, 1-4.
Hopp, P., Omer, M.K. and Heier, B.T. 2006. A case-control study of scrapie Nor98 in
Norwegian sheep flocks. J. Gen. Virol. 87, 3729-3736.

Hörnlimann, B., Bachmann, J. and Bradley, R. 2006. Portrait of bovine spongiform

encephalopathy in cattle and other ungulates. In Prions in Humans and Animals,
Eds. Hörnlimann, B., Riesner, D. and Kretschmar, H. Berlin: De Gruyter, pp:
233-249.

Hull, D.L. 1980. Individuality and selection. Annu. Rev. Ecol. Evol. 11, 311-332.

Hunter, N. and Bossers, A. 2007. The PrP genotype as a marker for scrapie
susceptibility. In: Prions in Humans and Animals, Eds. Hörnlimann, B., Riesner,
D. and Kretschmar, H. Berlin: De Gruyter, pp: 640-647.

Hurley, P.J. 2003. A Concise Introduction to Logic 8th Ed. Belmont CA:
Wadsworth/Thompson Learning.

Huxtable, C.R. and Dorling, P.R. 1982. Animal model of human disease,
mannosidosis, swainsonine-induced mannosidosis. Am. J. Path. 107, 124-126.

Jackson, M.P. and Hewitt, E.W. 2016. Cellular proteostasis: degradation of misfolded
proteins by lysosomes. Essays Biochem. 60, 173-180.

John, B., Hunter, C.A. and Harris, T.J. 2014. Immune Cell Trafficking in the Central
Nervous System. In Neuroinflammation and Neurodegeneration, Eds. P.K.
Peterson and Toborek, M. New York: Springer. pp: 29-45.

Jolly, R.D. and Walkley, S.U. 1997. Lysosomal storage diseases of animals: an essay
in comparative pathology. Vet. Pathol. 34, 527-548.

Jucker, M. and Walker, L.C. 2013. Self-propagation of pathogenic protein aggregates

in neurodegenerative diseases. Nature 501, 45–51.

29
Kessell, A.E. Finnie, J.W. and Windsor, P.A. 2011. Neurological diseases of ruminant
livestock in Australia. III: bacterial and protozoal infections. Aust. Vet. J. 89,
247-252.

Kimberlin, R.H. and Walker, C.A. 1978. Evidence that the transmission of one source
of scrapie agent to hamsters involves separation of agent strains from a mixture. J.
Gen. Virol. 39, 487-496.

Kipnis, J. 2016. Multifaceted interactions between adaptive immunity and the central
nervous system. Science 353, 766-71.

Kirschner, M. and Gerhart, J. 1998. Evolvability. Proc. Natl. Acad. Sci. U S A. 95,
8420-8427.

Konold, T., Davis, A., Bone, G., Bracegirdle, J., Everitt, S., Chaplin, M., Saunders,
G.C., Cawthraw, S. and Simmons, M.M. 2007. Clinical findings in two cases of
atypical scrapie in sheep: a case report. BMC Vet. Res. 3, 2.

Konold, T., Moore, S.J., Bellworthy, S.J. and Simmons, H.A. 2008. Evidence of
scrapie transmission via milk. BMC Vet. Res. 4, 14.

Konold, T., Moore, S.J., Bellworthy, S.J., Terry, L.A., Thorne, L., Ramsay, A.,
Salguero, F.J., Simmons, M.M. and Simmons, H.A. 2013. Evidence of effective
scrapie transmission via colostrum and milk in sheep. BMC Vet. Res. 9, 99.

Konold, T., Phelan, L.J., Clifford, D., Chaplin, M.J., Cawthraw, S., Stack, M.J. and
Simmons, M.M. 2014. The pathological and molecular but not clinical
phenotypes are maintained after second passage of experimental atypical bovine
spongiform encephalopathy in cattle. BMC Vet. Res. 10, 243.

Kroemer, G., Marino, G. and Levine, B. 2010. Autophagy and the integrated stress
response. Mol. Cell. 40, 280-293.

Lacroux, C., Corbiere, F., Tabouret, G., Lugan, S., Costes, P., Mathey, J., Delmas,
J.M., Weisbecker, J.L., Foucras, G., Cassard, H., Elsen, J.M., Schelcher, F. and
Andreoletti, O. 2007. Dynamics and genetics of PrPSc placental accumulation in
sheep. J. Gen. Virol. 88, 1056-1061.

Lacroux, C., Simon, S., Benestad, S.L., Maillet, S., Mathey, J., Lugan, S., Corbiere, F.,
Cassard, H., Costes, P., Bergonier, D., Weisbecker, J.L., Moldal, T., Simmons, H.,
Lantier, F., Feraudet-Tarisse, C., Morel, N., Schelcher, F., Grassi, J. and
Andreoletti, O. 2008. Prions in milk from ewes incubating natural scrapie. PLoS
Pathog. 4, e1000238.

LeGrand, E.K. and Brown, C.C. 2002. Darwinian medicine: applications of

30
 evolutionary biology for veterinarians. Can. Vet. J. 43, 556-559.

Lewontin, R.C. 1970. The Units of Selection. Annu. Rev. Ecol. Evol. 1, 1-18.

Li, J., Browning, S., Mahal, S.P., Oelschlegel, A.M. and Weissmann, C. 2010.
Darwinian evolution of prions in cell culture. Science 327, 869-872.

Ligios, C., Cancedda, G.M., Margalith, I., Santucciu, C., Madau, L., Maestrale, C.,
Basagni, M., Saba, M. and Heikenwalder, M. 2007. Intraepithelial and interstitial
deposition of pathological prion protein in kidneys of scrapie-affected sheep.
PLoS One 2, e859.
Ligios, C., Cancedda, M.G., Carta, A., Santucciu, C., Maestrale, C., Demontis, F.,
Saba, M., Patta, C., DeMartini, J.C., Aguzzi, A. and Sigurdson, C.J. 2011. Sheep
with scrapie and mastitis transmit infectious prions through the milk. J. Virol. 85,
1136-1139.

Linden, R. 2017. The biological function of the prion protein: a cell surface scaffold
of signaling modules. Front. Mol. Neurosci. 10, 77.

Louveau, A., Smirnov, I., Keyes, T.J., Eccles, J.D., Rouhani, S.J., Peske, J.D.,
Derecki, N.C., Castle, D., Mandell, J.W., Lee, K.S., Harris, T.H. and Kipnis, J.
2015. Structural and functional features of central nervous system lymphatic
vessels. Nature 523, 337-41.

Lühken, G., Buschmann, A., Brandt, H., Eiden, M., Groschup, M.H. and Erhardt, G.
2007. Epidemiological and genetical differences between classical and atypical
scrapie cases. Vet. Res. 38, 65-80.

M’Gowan, J.P. 1914. Investigation into the disease of sheep called “Scrapie”.
Edinburgh: William Blackwood and Sons.
(https://archive.org/details/b21272384).

Maddison, B.C., Baker, C.A., Rees, H.C., Terry, L.A., Thorne, L., Bellworthy, S.J.,
Whitelam, G.C. and Gough, K.C. 2009. Prions are secreted in milk from
clinically normal scrapie-exposed sheep. J. Virol. 83, 8293-8296.

Maddison, B.C., Rees, H.C., Baker, C.A., Taema, M., Bellworthy, S.J., Thorne, L.,
Terry, L.A. and Gough, K.C. 2010. Prions are secreted into the oral cavity in
sheep with preclinical scrapie. J. Infect. Dis. 201, 1672-1676.

Maestrale, C., Di Guardo, G., Cancedda, M.G., Marruchella, G., Masia, M., Sechi, S.,
Macciocu, S., Santucciu, C., Petruzzi, M. and Ligios, C. 2013. A lympho-
follicular microenvironment is required for pathological prion protein deposition
in chronically inflamed tissues from scrapie-affected sheep. PLoS One 8, e62830.

31
Mahal, S.P., Browning, S., Li, J., Suponitsky-Kroyter, I. and Weissmann, C. 2010.
Transfer of a prion strain to different hosts leads to emergence of strain variants.
Proc. Natl. Acad. Sci. U S A 107, 22653-22658.

Masujin, K., Shu, Y., Okada, H., Matsuura, Y., Iwamaru, Y., Imamura, M., Mohri, S.
and Yokoyama, T. 2009. Isolation of two distinct prion strains from a scrapie-
affected sheep. Arch. Virol. 154, 1929-1932.

Mayr, E. 1997. The objects of selection. Proc. Natl. Acad. Sci. USA 94, 2091–2094.

Mayr, E. 2001. What Evolution Is. London: Orion Books.

Mazza, M., Iulini, B., Vaccari, G., Acutis, P.L., Martucci, F., Esposito, E., Peletto, S.,
Barocci, S., Chiappini, B., Corona, C., Barbieri, I., Caramelli, M., Agrimi, U.,
Casalone, C. and Nonno, R. 2010. Co-existence of classical scrapie and Nor98 in
a sheep from an Italian outbreak. Res. Vet. Sci. 88, 478-485.

McEwen, B.S. and Wingfield, J.C. 2010. What’s in a name? Integrating homeostasis,
allostasis and stress. Horm. Behav. 57, 105.

McEwen, B.S., Bowles, N.P., Gray, J.D., Hill, M.N., Hunter, R.G., Karatsoreos, I.N.
and Nasca, C. 2015. Mechanisms of stress in the brain. Nat. Neurosci. 18, 1353-
1363.

McIntyre, K.M., del Rio Vilas, V.J. and Gubbins, S. 2008. No temporal trends in the
prevalence of atypical scrapie in British sheep, 2002-2006. BMC Vet. Res. 4,13.

McIntyre, K.M., del Rio Vilas, V.J. and Gubbins, S. 2008. No temporal trends in the
prevalence of atypical scrapie in British sheep, 2002-2006. BMC Vet. Res. 4, 13.

Montez, J.K. and Zajacova, A. 2013. Trends in mortality risk by education level and
cause of death among us white women from 1986 to 2006. Am. J. Public Health
103, 473-479.

Moore, S.J., Smith, J.D., Greenlee, M.H., Nicholson, E.M., Richt, J.A., and Greenlee,
J.J. 2016. Comparison of two US sheep scrapie isolates supports identification as
separate strains. Vet. Pathol. 53, 1187-1196.

Morales, R., Hu, P.P., Duran-Aniotz, C., Moda, F., Diaz-Espinoza, R., Chen, B.,
Bravo-Alegria, J., Makarava, N., Baskakov, I.V. and Soto, C. 2016. Strain-
dependent profile of misfolded prion protein aggregates. Sci. Rep. 6, 20526.

Moum, T., Olsaker, I., Hopp, P., Moldal, T., Valheim, M., Moum, T. and Benestad,
S.L. 2005. Polymorphisms at codons 141 and 154 in the ovine prion protein gene
are associated with scrapie Nor98 cases. J. Gen. Virol. 86, 231-235.

32
National Academies of Sciences, Engineering, and Medicine. 2017. Approaches to
Understanding the Cumulative Effects of Stressors on Marine Mammals. The
National Academies Press: Washington, DC.

Nesse, R.M., Bergstrom, C.T., Ellison, P.T., Flier, J.S., Gluckman, P., Govindaraju,
D.R., Niethammer, D., Omenn, G.S., Perlman, R.L., Schwartz, M.D., Thomas,
M.G., Stearns, S.C. and Valle, D. 2010. Evolution in health and medicine Sackler
colloquium: Making evolutionary biology a basic science for medicine. Proc.
Natl. Acad. Sci. U S A. 107 Suppl 1, 1800-1807.

OIE. 2017a. Scrapie, Chapter 2.7.12. In Manual of Diagnostic Tests and Vaccines for
Terrestrial Animals 2017. Paris: World Organisation for Animal Health.

OIE. 2017b. Veterinary Services. Chapter 3.1. In OIE Terrestrial Animal Health Code.
Paris: World Organisation for Animal Health.

Onodera, T., Ikeda, T., Muramatsu, Y. and Shinagawa, M. 1993. Isolation of scrapie
agent from the placenta of sheep with natural scrapie in Japan. Microbiol.
Immunol. 37, 311-316.

Orge, L., Oliveira, A., Machado, C., Lima, C., Ochoa, C., Silva, J., Carvalho, R.,
Tavares, P., Almeida, P., Ramos, M., Pinto, M.J. and Simas, J.P. 2010. Putative
emergence of classical scrapie in a background of enzootic atypical scrapie. J.
Gen. Virol. 91, 1646-1650.

Ortiz-Pelaez, A., Arnold, M.E. and Vidal-Diez, A. 2016. Epidemiological

investigations on the potential transmissibility of a rare disease: the case of
atypical scrapie in Great Britain. Epidemiol. Infect. 144, 2107-2116.

Parham, P. 2014 The Immune System. 4th Ed. New York: Garland Science.

Parry, H.B. 1966. Natural (spontaneous) scrapie in sheep. I. Clinical manifestation

and general incidence, treatment, and related syndromes. In Report of Scrapie
Seminar Held At Washington, D.C., January 27-30, 1964. Agricultural Research
Service, US Department of Agriculture. pp. 95-103.
https://archive.org/stream/report9153scra/report9153scra_djvu.txt.

Parry, H.B. 1983. Scrapie Disease in Sheep: Historical, Epidemiological, Pathological

and Practical Aspects of the Natural Disease. Ed. Oppenheimer, D.R. London:
Academic Press.

33
Pattison, I.H. and Millson, G.C. 1961. Scrapie produced experimentally in goats with
special reference to the clinical syndrome. J. Comp. Pathol. 71, 101-109.

Pattison, I.H., Hoare, M.N., Jebbett, J.N. and Watson. W.A. 1972. Spread of scrapie
to sheep and goats by oral dosing with foetal membranes from scrapie-affected
sheep. Vet. Rec. 90, 465-468.

Pattison, I.H., Hoare, M.N., Jebbett, J.N., Watson, W.A. 1974. Further observations
on the production of scrapie in sheep by oral dosing with foetal membranes from
scrapie-affected sheep. Br.Vet. J.130, lxv-lxviii.

Perrott, M.R., Sigurdson, C.J., Mason, G.L. and Hoover, E.A. 2012. Evidence for
distinct chronic wasting disease (CWD) strains in experimental CWD in ferrets. J.
Gen. Virol. 93, 212-221.

Prusiner, S.B. 2013. Biology and genetics of prions causing neurodegeneration. Ann.
Rev. Genet. 47, 601–623.

Race, R., Jenny, A. and Sutton, D. 1998. Scrapie infectivity and proteinase K-resistant
prion protein in sheep placenta, brain, spleen, and lymph node: implications for
transmission and antemortem diagnosis. J. Infect. Dis. 178, 949-953.

Radostits, O.M., Gay, C.C., Blood, D.C. and Hinchcliff, K.W. 2000. Veterinary
Medicine: A Textbook of the Diseases of Cattle, Sheep, Pigs, Goats and Horses.
London: W.B. Saunders Company Ltd.

Ransohoff, R. 2016. How neuroinflammation contributes to neurodegeneration.

Science 353, 777-783.

Ransohoff, R.M. and Engelhardt, B. 2012. The anatomical and cellular basis of
immune surveillance in the central nervous system. Nat Rev Immunol. 12. 623-
35.
Rothman, K.J. and Greenland, S. 2005. Causation and causal inference in
epidemiology. Am. J. Public Health 95, Suppl. 1, S144-50.

Rubenstein, R., Bulgin, M.S, Chang, B., Sorensen-Melson, S., Petersen, R.B. and
Lafauci, G. 2012. PrPSc detection and infectivity in semen from scrapie-infected
sheep. J. Gen Virol. 93,1375-1383.

Rubenstein, R., Chang, B., Gray, P., Piltch, M., Bulgin, M.S., Sorensen-Melson, S.
and Miller, M.W. 2011. Prion disease detection, PMCA kinetics, and IgG in urine
from sheep naturally/experimentally infected with scrapie and deer with
preclinical/clinical chronic wasting disease. J. Virol. 85, 9031-9038.

34
Salazar, E., Monleon, E., Bolea, R., Acin, C., Perez, M., Alvarez, N., Leginagoikoa, I.,
Juste, R., Minguijon, E., Reina, R., Glaria, I., Berriatua, E., de Andres, D.,
Badiola, J.J., Amorena, B. and Lujan, L. 2010. Detection of PrPSc in lung and
mammary gland is favored by the presence of Visna/maedi virus lesions in
naturally coinfected sheep. Vet. Res. 41, 58.

Sandberg, M. K., Al-Doujaily, H., Sharps, B., Clarke, A. R. and Collinge, J. 2011.
Prion propagation and toxicity in vivo occur in two distinct mechanistic phases.
Nature 470, 540-542.

Schneider, K., Fangerau, H., Michaelsen, B., Raab, W.H. 2008. The early history of
the transmissible spongiform encephalopathies exemplified by scrapie. Brain Res.
Bull. 77, 343-355.

Solforosi, L., Milani, M., Mancini, N., Clementi, M., Burioni, R. 2013. A closer look
at prion strains: characterization and important implications. Prion 7, 99-108.

Sontag, E.M., Samant, R.S. and Frydman, J. 2017. Mechanisms and functions of
spatial protein quality control. Annu. Rev. Biochem. 8, 97-122.

Spiropoulos, J., Hawkins, S.A.C., Simmons, M.M. and Bellworthy, S.J. 2014.
Evidence of in utero transmission of classical scrapie in sheep. J. Virol. 88, 4591-
4594.

Terry, L. A., L. Howells, K. Bishop, C. A. Baker, S. Everest, L. Thorne, B. C.

Maddison, and Gough K. C. 2011. Detection of prions in the faeces of sheep
naturally infected with classical scrapie. Vet. Res. 42, 65.

Thackray, A. M., Hopkins, L., Klein, M. A. and Bujdoso, R.

2007. Mouse-adapted ovine scrapie prion strains are characterized by different
conformers of PrPSc. J. Virol. 81, 12119-12127.

Thackray, A. M., Hopkins, L., Lockey, R., Spiropoulos, J. and Bujdoso, R.

2011. Emergence of multiple prion strains from single isolates of ovine scrapie. J.
Gen. Virol. 92, 1482-1491.

Thackray, A. M., Hopkins, L., Spiropoulos, J. and Bujdoso, R.

2008. Molecular and transmission characteristics of primary-passaged ovine scrapie
isolates in conventional and ovine PrP transgenic mice. J. Virol. 82:11197-11207.

Thackray, A. M., Lockey, R., Beck, K. E., Spiropoulos, J. and Bujdoso, R.

2012. Evidence for co-infection of ovine prion strains in classical scrapie isolates. J.
Comp. Pathol. 147, 316-329.

Tsigios, C., Kyrou, I., Kassi, E. and Chrousos, G.P. 2016. Stress, Endocrine

35
 Physiology and Pathophysiology. In Endotext [Internet], Eds. De Groot, L.J.,
Chrousos, G., Dungan, K., Feingold, K.R., Grossman, A., Hershman, J.M., Koch,
C., Korbonits, M., McLachlan, R., New, M., Purnell, J., Rebar, R., Singer, F. and
Vinik, A. South Dartmouth (MA): MDText.com, Inc.

Tuo, W., O’Rourke, K.I., Zhuang. D., Cheevers, W.P., Spraker, T.R. and Knowles,
D.P. 2002. Pregnancy status and fetal prion genetics determine PrPSc
accumulation in placentomes of scrapie-infected sheep. Proc. Natl. Acad. Sci. U
S A 99, 6310-6315.

Tuo, W., Zhuang, D., Knowles, D.P., Cheevers, W.P., Sy, M.S. and O’Rourke, K.I.
2001. Prp-c and Prp-Sc at the fetal-maternal interface. J. Biol. Chem. 276, 18229-
18234.
Ulvund, M. J. 2006. Clinical Findings in Scrapie. In Prions in Humans and Animals,
Eds. Hörnlimann, B., Riesner, D. and Kretzschmar. Berlin: de Gruyter, pp. 398-
407.

Ulvund, M. J. 2008. Ovine scrapie disease: do we have to live with it? Small Rumin.
Res. 76, 131 - 140.

Uptain, S.M. and Lindquist, S. 2002. Prions as protein-based genetic elements. Annu
Rev Microbiol 56, 703-741.

Van Keulen, L.J., Bossers, A., van Zijderveld, F. 2008. TSE pathogenesis in cattle
and sheep. Vet Res 39, 24.

Vascellari, M., Nonno, R., Mutinelli, F., Bigolaro, M., Di Bari, M.A., Melchiotti, E.,
Marcon, S., D’Agostino, C., Vaccari, G., Conte, M., De Grossi, L., Rosone, F.,
Giordani, F. and Agrimi, U., 2007. PrPSc in salivary glands of scrapie-affected
sheep. J. Virol. 81, 4872-4876.

Vulin, J., Beck, K.E., Bencsik, A., Lakhdar, L., Spiropoulos, J. and Baron, T. 2012.
Selection of distinct strain phenotypes in mice infected by ovine natural scrapie
isolates similar to CH1641 experimental scrapie. J. Neuropathol. Exp. Neurol. 71,
140-147.

Walker, L.C. and Jucker, M. 2015. Neurodegenerative diseases: expanding the prion
concept. Annu. Rev. Neurosci. 38, 87-103.

Watts J.C., Giles, K., Patel, S., Oehler, A., DeArmond, S.J. and Prusiner, S.B. 2014.
Evidence that bank vole PrP is a universal acceptor for prions. PLoS Pathog. 10,
e1003990.

Weller, R.O., Djuanda, E., Yow, H-Y. and Carare, R.O. 2009. Lymphatic drainage of
the brain and the pathophysiology of neurological disease. Acta. Neuropathol.

36
 117, 1–14.

Wemheuer, W.M., Benestad, S.L., Wrede, A., Wemheuer, W.E., Brenig, B., Bratberg,
B. and Schulz-Schaeffer, W.J. 2011. PrPSc spreading patterns in the brain of
sheep linked to different prion types. Vet. Res. 42, 32.

Wenborn, A., Terry, C., Gros, N., Joiner, S., D’Castro, L., Panico, S., Sells, J.,
Cronier, S., Linehan, J.M., Brandner, S. and Saibil, H.R. 2015. A novel and rapid
method for obtaining high titre intact prion strains from mammalian brain. Sci.
Rep. 5,10062.

Wickner, R.B., Edskes, H.K., Roberts, B.T., Baxa, U., Pierce, M.M., Ross, E.D. and
Brachmann, A. 2004. Prions: proteins as genes and infectious entities. Genes Dev.
18, 470-485.

Wilkinson, R. and Marmot, M. 2003. Social Determinants of Health; The Solid Facts.
2nd Ed. WHO Regional Office for Europe: World Health Organisation.

Williams, E.S. 2005. Chronic wasting disease. Vet. Pathol. 42, 530-549.

Wilson, D.R., Anderson, R.D. and Smith, W. 1950. Studies in scrapie. J. Comp.
Pathol. 60, 267-282.

Wraith, D.C. and Nicholson, L.B. 2012. The adaptive immune system in diseases of
the central nervous system. J. Clin. Invest. 122, 1172-1179.

Wulf, M.A., Senatore, A. and Aguzzi, A. 2017. The biological function of the cellular
prion protein: an update. BMC Biol. 15, 34.

Yokoyama, T., Masujin, K., Schmerr, M. J., Shu, Y., Okada, H., Iwamaru, Y.,
Imamura, M., Matsuura, Y., Murayama, Y. and Mohri, S. 2010. Intraspecies
prion transmission results in selection of sheep scrapie strains. PLoS One 5,
e15450.

37
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