Open Vets. 2020; 1: 1-14

Review Article

David B. Adams* Evolvability and the Hazard of Atypical Scrapie in Small Ruminants https://doi.org/10.1515/ovs-2020-0001 received April 22, 2019; accepted August 20, 2019 1 Introduction

The discipline of evolutionary biology provides special Abstract: Observations on strain behaviour and direct insights into the natural history of and is vital demonstrations of natural selection establish that the to the practice of and public health across scrapie agent and in general are able to evolve. all species [1, 2]. Evolutionary biology has been applied Accordingly, it is conceivable that atypical non-conta- to scrapie disease in [3] where it showed that the gious scrapie in sheep and can transform to classi- scrapie agent, a prion or ‘proteinaceous infectious agent’ cal contagious scrapie under particular circumstances. In [4], and prions in general possess the functions of varia- consequence, atypical scrapie can be regarded as a latent tion, reproduction and heritability that drive evolution [5]. hazard that warrants comprehensive risk assessment Prions are thus endowed with evolvability or the capac- and biosecurity preparedness planning. Evidence for this ity for generating adaptive diversity and evolving through proposition comes from differences in the expression of natural selection [6, 7] and can be regarded as objects atypical and classical scrapie that may make scrapie con- of selection [8, 9]. The extended evolutionary synthesis tagious, historical records of scrapie in Western , [10] is significant in this regard. It caters for evolution in and contemporary accounts of the epidemiology of atyp- the absence of a nucleic acid based genome and allows ical scrapie. Biosecurity preparedness can be based on for evolutionary mechanisms involving prions or pro- current knowledge of pathophysiology and epidemiol- teins that acquire alternative conformations that become ogy and can be built around a three-stage model for the self-propagating [11]. endogenous emergence of a propagating epidemic of The evolvability of prions is substantiated by strain scrapie. The first stage concerns the occurrence of atypi- behaviour in the scrapie agent and has important prac- cal scrapie. The second stage concerns the acquisition of tical consequences for scrapie in sheep [3]. It justifies communicability in prion populations provided by atyp- the proposition that non-contagious idiopathic forms of ical scrapie and the third stage concerns circumstances scrapie, represented arbitrarily by atypical scrapie [12] can allowing disease and the initiation of a be the precursor of classical contagious scrapie. Accord- propagating epidemic. The range of component causes ingly, atypical scrapie is identified as a latent hazard that envisaged for possible outbreaks of endogenous classical can convert to a contagious form under particular circum- scrapie is broad. However, exposure of sheep and goats to stances. The question now is what these circumstances cyanobacterial toxins qualifies for special attention. might be and how they might be managed. The present study reviews relevant knowledge on the natural history of scrapie and the known behaviour of Keywords: Proteostasis; Biosecurity; Neurotoxin; Neuro- prions in the context of evolutionary biology and patho- degenerative physiology. The objective is a preliminary understanding of factors involved in the possible endogenous emergence of classical contagious scrapie from atypical non-conta- gious scrapie and the intent is to inform risk management. Accordingly, (1) differences between atypical and classical scrapie are recapitulated, (2) possible cellular and molec- ular mechanisms underlying the idiopathic genesis of *Corresponding author: David B. Adams, 24 Noala Street, Aranda, atypical scrapie are investigated, (3) the historical record ACT 2614, , Tel: +61 2 6161 4825, E-mail: dadams@home- of scrapie disease in sheep is probed for possible inde- mail.com.au pendent appearances of scrapie at different places and

Open Access. © 2019 David B. Adams., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 License. 2 D. B. Adams different times, and (4) accounts of the epidemiological tests based on immunoblotting and immunohistochem- aspects of atypical and Nor98 strain scrapie are analysed. istry. This work used an outstanding collection of case histories to classify the clinical expression of scrapie into five typical syndromes of classical scrapie and five atypi- cal syndromes of scrapie-like illness in sheep. More recent 2 Differences between atypical and studies in 2006 and 2008 [16, 21] list 33 different clini- classical scrapie cal signs of scrapie from four different scientific reports and clustered them under headings of ‘general signs’, A consensus view from the Panel on Biological Hazards ‘changes in behaviour’, ‘changes in sensitivity’, ‘changes of the European Food Safety Authority states that atypical in locomotion’ and ‘other signs’. Symptomatology varied scrapie ‘does not present, epidemiologically, like an infec- according to place and sheep population. Altered mental tious disease’ [12] (EFSA, 2014). A more recent review [13] status, pruritus and wool loss were the most frequent of scrapie disease states similarly that atypical scrapie has initial signs seen in Norway and Spain whereas ataxia a separate aetiology from classical scrapie and is “sponta- and head tremours were the most frequent initial seen in neous or transmits very poorly under natural conditions”. Ireland and both ataxia and pruritis were seen in Italy. Unlike classical scrapie, atypical scrapie is not listed by Characteristics of classical scrapie and atypical scrapie the World Organization for Animal Health (OIE) as a noti- that differ in kind as opposed to degree are the nature and fiable disease [14]. distribution of lesions and deposits of deformed or mis- For present purposes, atypical scrapie is postulated folded prion (PrPSc ) in the central , as non-contagious scrapie and its comparison with con- the distribution of infectivity and misfolded prion tagious classical scrapie can identify features associated protein (PrPSc) and the immunoblot pattern. These first with the capability for contagion. In this regard, future two differences may point to mechanisms that are present instances where atypical scrapie is found to be contagious in classical scrapie and which are required for atypical will not refute the possibility of non-contagious scrapie scrapie to become contagious. The significance of the or the cogency of earlier accounts of atypical scrapie. third difference, the presence of a fourth band of 15 kDa in Instead, they will fortify the present thesis that atypical the immunoblot pattern seen atypical scrapie, is unclear. scrapie has a potential for transforming to contagious As to the first difference, there can be no contagion scrapie when particular circumstances apply. if prions are restricted to the central nervous system and A review of key aspects of classical and atypical thus have no access to the portals of exit known to be scrapie, including Nor98 scrapie [15], shows that some involved in the transmission of contagious scrapie. In characteristics in classical compared with atypical scrapie this regard, PrPSc has been found in the brain of sheep differ in degree whereas other characteristics differ in with atypical scrapie but not in peripheral tissues [22, 23]. kind [3]. Differences in degree may reflect the diversity of By contrast, PrPSc is found in both brain and peripheral variants within population of prions involved in classical tissues in sheep with classical scrapie [24-26]. Particular- and atypical scrapie and point to scope for evolutionary ities in the nature and distribution of lesions and in the change. Differences in kind, however, may foreshadow central nervous system of sheep with atypical or classical gains-of-function in prion populations that can allow the scrapie [27, 28] may identify portals of exit of the scrapie conversion of non-contagious atypical scrapie to conta- agent to peripheral tissues and suggest possible patho- gious scrapie. physiological mechanisms for the migration of scrapie Characters of classical scrapie and atypical scrapie prions to the rest of the body. that differ in degree as opposed to kind are age of onset The second difference in kind between atypical and of disease, symptomatology and duration of disease. classical scrapie is also relevant to the portals of exit that The tendency for atypical scrapie to occur in older sheep make scrapie contagious. The presence of PrPSc and infec- [16-18] may reflect the aetiological importance of and tivity in the lymphoreticular tissues and cells is a constant environment rather than exposure to exogenous infec- feature in sheep with contagious classical scrapie [24- tion, which drives classical scrapie. Classical and atypical 26]. In contrast, PrPSc is virtually restricted to the central scrapie have exceptionally diverse signs and symptoms nervous system and is scant in the lymphoreticular that vary according to place, time and population and tissues and cells of sheep with atypical scrapie [22, 28]. signal the extent of variation in the scrapie agent. For This difference is crucial because the well-known migra- example, the work of Parry [19, 20], carried out from 1950 tory behaviour of lymphocytes and mononuclear phago- to 1980 in England, predates the availability of diagnostic cytes [29] provides a means for disseminating the scrapie Prion Evolvability and the Hazard of Atypical Scrapie in Small Ruminants 3 agent throughout the body and to the multiple portals of influence the kinetics of PrPSc accumulation as follows: exit that operate either prenatally or post-natally [30]. It (1) is intrinsically error-prone [35] and is noteworthy that highly sensitive transmission studies an induced demand for PrPC may produce an amount of with transgenic mice over-expressing the VRQ allele of PrPSc that exceeds the adaptive capacity of proteasomes the ovine prion protein gene have detected infectivity in and lysosomes. (2) An induced increase in the rate of PrPC lymphoid tissues, nerves, and muscles in natural cases of misfolding may exceed the adaptive capacity of clear- atypical/Nor 98 scrapie and also in atypical/Nor98 scrapie ance mechanisms in proteasomes and lysosomes. (3) induced by intracerebral inoculation [23]. This indicates a An induced decrease in the capacity of clearance mech- germinal or latent capacity within the prions of atypical anisms in proteasomes and lysosomes may lead to a scrapie to translocate throughout the body and to access build-up of PrPSc produced at an otherwise ordinary rate. known portals of exit. (4) An induced increase in the production of PrPSc may coincide with an induced decrease in the capacity of clear- ance mechanisms in proteasomes and lysosomes. (5) The situation may be chaotic with unregulated fluctuations in 3 Cellular and molecular mecha- the production and misfolding of PrPC and the clearance nisms involved in the genesis of of PrPSc. The causal factors responsible for impaired prote- atypical scrapie ostasis and the cellular abnormalities that lead to the emergence of atypical scrapie may be genetic or acquired, The functional failure at the root of all forms of scrapie or a combination of the two. The pathways involved in and other prion disease relates to a structurally abnormal the lysosomal degradation of cytoplasmic organelles or form of the cellular prion protein (PrPC) that foreshadows cytosolic components can be affected by fluctuations in harm in the cells, tissues, organs and the body as a whole. a broad range of external conditions [37]. These include Variations in the clinical expression of disease will result physical factors (such as temperature and ultraviolet from variations in the structurally abnormal form of the light), chemical factors (such as ion concentrations, pH, cellular prion protein (PrPC). Abnormal prions (PrPSc) oxygen tension, redox potentials and metabolite concen- and their toxic intermediates [31] originate either exoge- trations), extracellular signals (such as contact-depend- nously as in classical scrapie or endogenously as in atyp- ent signals, hormones, cytokines and neurotransmitters) ical scrapie and proliferate by means of seeded protein and microbial . It is proposed that when these polymerization in which a PrPSc seed binds with PrPC and factors exceed a certain threshold they can be considered acts as a template for misfolding [32]. The resulting poly- ‘stresses’ that determine the function and survival of cells mers split to produce more seeds [33, 34]. Scrapie disease [37]. reflects defective protein homeostasis and comes into If the possibilities outlined for impaired proteostasis existence when rates of PrPSc proliferation exceed rates of [35] are extended to the genesis of atypical scrapie, vir- PrPSc clearance and there is an accumulation of PrPSc and tually nothing within sheep or their environment can be toxic intermediates. Cellular prion protein is the product excluded as a causal factor. Best protection against the of the highly conserved prion gene (PRNP) and there are emergence of atypical scrapie may occur through manage- no theoretical grounds for proposing that variants of this ment to optimise the health and wellbeing of sheep and gene may produce a protein product that is insusceptible thus to optimise their capacity to cope with stressors and to misfolding. recover from the impacts of stress. Figure 1 sets out a general model for the cellular life Indolizidine alkaloids are worthy of mention as one cycle of the scrapie agent in atypical and classical scrapie of many possible hazards for the emergence of atypical and shows factors involved in the proliferation and clear- scrapie. These alkaloids include swainsonine, which ance of abnormal prions (PrPSc). As for all , the inhibits an in lysosomes (α-D-mannosidase), cellular prion protein (PrPC) has a half-life and mecha- leads to vacuolation in the nervous system, and produces nisms for protein homeostasis that operate through the a neurological syndrome in affected animals [38]. Swain- proteostasis network [35, 36] produce a dynamic equilib- sonine is the poisonous principle produced by endophytic rium between synthesis and degradation that harmonises fungi in leguminous plants such as Swainsona spp. in Aus- with changing demands for the prion protein. tralia, Astralagus spp. in and Oxytropis spp. Various factors related to protein biosynthesis and in China and can lead to considerable losses in grazing protein clearance by proteasomes and lysosomes may animals such as sheep [39]. It is relevant that swainsonine 4 D. B. Adams

Figure 1: Pathways for the production and removal of functional PrPC and pathways for the misfolding and subsequent turnover of dys- functional prions (PrPSc). Biosynthesis of normal cellular prion protein (PrPC) occurs in the ribosome and subsequent folding is assisted by molecular proteins. Points where impairments to the proteostasis network may influence the kinetics of PrPSc accumulation are indicated. has been used to demonstrate the mutation and selection 4 Historical accounts of classical of prion strains in neuroblastoma culture and action of evolutionary forces [40]. An analysis of the - scrapie in sheep and their implica- esis involved in other plant-derived, fungal-derived and tions for atypical scrapie cyanobacterial-derived poisonings could point to other possibilities for the onset of atypical scrapie. Cyanobac- Parry [20] stated that the clinical forms and epidemiolog- terial toxins are highlighted because of the strong causal ical characteristics of scrapie in sheep are such that “it link to neurodegenerative in people [41-45]. is not mere chance that has ensured an extremely high standard in the accounts of the disease published in many Prion Evolvability and the Hazard of Atypical Scrapie in Small Ruminants 5 parts of north-western Europe from 1750 onwards”. As a banded and bred in relative isolation. Significantly, geo- consequence, histories of scrapie published in 1914 by graphic distributions of some scrapie-free breeds over- McGowan [46], in 1983 by Parry [20] and in 2008 by Sch- lapped those of scrapie-affected breeds, implying that neider et al. [47] can shed light on the pattern of occur- agent-related and environment-related causative factors rence of scrapie according to time, place and population had minimal influence. Nineteen breeds are recorded with of sheep. In doing so, these histories can assist in inves- scrapie and fifteen breeds are recorded with no scrapie. In tigating the proposition that atypical scrapie is prospec- breeds such as the Norfolk Horn, Wiltshire Horn, Dorset tive classical scrapie and in understanding the causative Horn, and Hampshire Down, scrapie appeared in reports factors that may allow the possibility of emergence. between 1750 and 1800, disappeared for a century and The question put to the history of scrapie is whether reappeared after 1900. In breeds such as the Welsh Moun- the disease has appeared or reappeared in different popu- tain, Herdwick, Teeswater, Scottish Blackface, Shetland, lations of sheep at different times and in different places. Oxford Down and Suffolk, scrapie was not recorded until Have outbreaks of scrapie in sheep populations always after 1900. The pattern of scrapie occurrence according to arisen through incursions of the scrapie agent from time, breed and place was unlike the pattern displayed by disease reservoirs in other populations? In other words, to other diseases of sheep and suggests a major influence what extent can recorded occurrences of scrapie be attrib- from host-related causative factors. The Spanish merino uted to the migration of infected animals or the movement may have been important in outbreaks of scrapie in France of fomites or does the pattern of outbreaks signal endog- and Germany but can be discounted as a cause of the mul- enous emergence of the disease and a new reservoir of tiple and geographically separated occurrences of scrapie the scrapie agent? The accounts of M’Gowan [46], Parry seen in Great Britain before 1787 when the Spanish merino [20] and Schneider et al. [47] collate secondary and ter- first entered that country. tiary historical sources and do not build from first hand M’Gowan [46] cites eight references that describe records. As for all history, these accounts will not provide occurrences of scrapie in sheep in Germany and France. definitive answers to questions posed. However, they give Scrapie is reported in Upper Saxony before 1800 and three a crude impression of the past where the general tenden- references speak to the existence of scrapie in France cies of scrapie can be gauged according to the epidemi- during the 1800s. M’Gowan reproduced in full an account ological triangle of host, disease agent and environment of scrapie in Germany that was published in 1868 by Georg [48]. May. Parry [19] tabulates information on the prevalence of M’Gowan [46] in 1914 dwells upon the various names scrapie in France, Germany and the Danube Valley from given to scrapie in Great Britain (e.g. scratchie, rubbers, 1700 to 1980. In France, scrapie is reported as severe in rickets, goggles, shakings, shrewcroft and cuddie trot) merinos at Rambouillet between 1780 and 1820, moderate and refers to the disease in south-western and south-east- but widespread (and extending to milk sheep) between ern England, northernmost England and southernmost 1820 and 1880, and as sporadic outbreaks in many areas Scotland. Scrapie, named as ‘rickets’, was reported in Lin- between 1880 and 1950. In Germany, scrapie is reported colnshire before 1750. Between 1750 and 1800, scrapie was as being present perhaps between 1700 and 1750, occur- reported in Cambridgeshire, Huntingdonshire, Lincoln- ring in merinos in Saxony and Silesia between 1750 and shire, Norfolkshire, Essex, Kent and Sussex in south-east- 1780, occurring as epidemics in Brandenburg and Saxony ern England, in Hampshire, Wiltshire, Dorsetshire and between 1780 and 1820, being severe in Electoral merinos Somerset in south-western England and in Lancashire in the east but not the south between 1820 and 1880, and in the northern sector of England. Scrapie was reported declining then becoming rare and very rare between 1880 in Devonshire and Berkshire between 1800 and 1850 and and 1950. Reference is made to an outbreak in 1973 of in Northumberland and Roxburghshire between 1850 scrapie in merinos in eastern Germany. As to the Danube and 1900. The only breed mentioned by M’Gowan [46] is Valley, scrapie is reported as severe in Electoral merinos the Wiltshire and inbreeding is referred to as a causative between 1820 and 1880 then declining to none after 1930. factor for scrapie. Outbreaks of scrapie occurred after 1950 in Hungary Parry [20] extends the history of scrapie in Great and Bulgaria and can be attributed to the scrapie agent Britain to the end of the 1970s and presents observations present in Suffolks imported from England. In like manner, according to breed of sheep, derivation of the breed, time earlier outbreaks of scrapie in merinos, particularly periods of 50 years, geographical location and an arbi- Electoral merinos and their descendants, in France and trary ordinal grade of scrapie occurrences. Breeds provide Germany, can be attributed to incursions of the scrapie a useful indicator of particular populations of sheep hus- agent. However, Georg May in Germany in 1868 does not 6 D. B. Adams accept this as the whole explanation. May delved deeper first report in Norway [49] concluded that scrapie Nor98 and connected the outbreaks of scrapie in question to a differed from classical scrapie in its distribution pattern list of possible ‘immediate’ and ‘remote’ causes related to and had a low to null communicability in the field. Sub- imprudent breeding practices and adverse conditions of sequent reports have drawn similar conclusions with husbandry. Importantly, occurrences of classical scrapie none giving any hint that scrapie Nor98 is infectious. For in Great Britain in the early eighteenth century occurred example, analysis of the records of active surveillance in the absence of merinos and point to separate endog- for transmissible spongiform in small enous origins of the disease. This observation provides ruminants in eleven European countries between 2004 grounds for claiming that similar separate endogenous and 2007 [51] prompted a conclusion that ‘atypical scrapie events may explain some of the outbreaks of classical is not contagious or has a very low transmissibility under scrapie in France and Germany during the eighteenth and natural conditions compared with classical scrapie’. nineteenth centuries. Several studies highlight the sporadic nature of atypical Schneider et al. [47] in 2008 outline the early litera- scrapie Nor98 [17, 49, 52, 53] and its resemblance to spo- ture on the transmissible spongiform encephalopathies radic Creutzfeldt–Jakob disease in . and explore viewpoints about the causes and modes of In 2007, Lühken et al. [17] described differences in the transmission of scrapie. Possible causes contemplated geographic-distribution, age-distribution and breed-dis- during the 1800s and late 1700s included factors in the tribution of atypical and classical scrapie. Atypical scrapie physical environment (thunderstorms, other exceptional occurred across the whole of Germany whereas classical atmospheric events, humidity), nutritional environment scrapie was restricted to southern Germany. Classical (excessive or inadequate feeding, sodium or potassium scrapie was not observed in sheep over seven years old deficiency), biotic environment (e.g. ‘worm’ infection) and whereas 43% of cases of atypical scrapie were found in husbandry environment (breeding from sheep too young sheep over seven years old. Classical scrapie was recorded or too old, cross-breeding, in-breeding and tail docking). in three breeds and in crossbreeds whereas atypical The most common view, however, was that host-related scrapie was found in eleven of twelve breed categories. factors (heredity) were paramount in occurrences of clas- Peculiarities of genetics were also observed with the ARQ/ sical scrapie. ARQ PRNP genotype being most susceptible to classical

In sum, the histories offered by Parry [20], M’Gowan scrapie and the AF141RQ and AHQ haplotype having the [46] and Schneider et al. [47] indicate that classical scrapie highest risk for atypical scrapie. in Great Britain and continental Europe appeared at dif- Epidemiological investigations into possible determi- ferent times and places as discrete episodes that were gen- nants or component causes of atypical scrapie are availa- erated by factors related to sheep and their environment ble for sheep populations in Norway [49], France [56] and and not by incursions of scrapie from outside sources. Great Britain [53]. In Norway [49], there was an increased The pattern observed is consistent with a sporadic form risk of atypical scrapie Nor98 in flocks given vitamin and of scrapie transforming to a communicable form and then mineral feed supplements, which could suggest either a to a full-blown epidemic under the influence of three sets causative role for certain vitamins or minerals or the effect of factors acting in sequence (shown later in Figure 2). The of factors that called for feed supplementation. In France first set of factors would affect the occurrence of sporadic [56], sheep dairy farms had a higher risk for atypical scrapie. The second set would affect the acquisition of scrapie, which may reflect particular exposures such as communicability and create the prospect of a propagating metabolic disorders related to lactation. In contrast to the epidemic. The third set of factors would allow transmis- findings in Norway [49], the feeding of vitamin and mineral sion and initiate a propagating epidemic of scrapie. supplements was associated with a lower risk of atypical scrapie, which may be explained by differing environmen- tal conditions and differing interactions between diet, mineral intake and local stressors. In France [56], sheep 5 Epidemiological studies on on farms operating under guidelines for organic produc- atypical scrapie -Nor98 in sheep tion and sheep fed corn silage had lower risks for atypi- cal scrapie, which was attributed to ‘less harsh farming The incidence and distribution of atypical scrapie has conditions’. In Great Britain [53], the status of sheep farms been investigated in sheep populations in Norway [49], as ‘organic’ had no significant impact on the risk of atyp- Germany [16], Great Britain [50, 52-54], Wales [55], France ical scrapie. Breed had a significant impact on the risk of [56, 57] , and in multiple European countries [18, 51]. The atypical scrapie with Welsh Mountain and Cheviot breeds Prion Evolvability and the Hazard of Atypical Scrapie in Small Ruminants 7 strongly associated presence of the disease and Charolais which involve prions or ‘proteinaceous nucleating parti- sheep associated with absence of the disease. Studies in cles’ [63, 64]. Great Britain [53] also found that the greater the number Historical accounts of scrapie [20, 46, 47] indicate of visits by veterinarians the smaller the odds of atypical that propagating epidemics of the disease can have new scrapie. Some explanations were offered but the simplest individual beginnings at different times and places and in may be a connection between the use of veterinary ser- different populations of sheep. In doing so they endorse vices and diligence regarding animal health and wellbe- the notion that atypical scrapie can give rise to classical ing. scrapie. Occurrences of scrapie in Great Britain before Information about the multitude of possible determi- the introduction of stocks of merinos in 1787 are crucial. nants of atypical scrapie may be rudimentary. However, They show that the origins of scrapie need not be uniquely all listed accounts of atypical scrapie according to occur- attributed to the merino breed as suggested by experi- rences in time, place and population indicate that cases ence with scrapie in eighteenth and nineteenth century occur without transmission from sheep to sheep or France and Germany where outbreaks were associated between flocks. Atypical scrapie can be characterised as a with merinos (particularly Electoral merinos) and their ‘spontaneous disease with a genetic determinant and pos- descendants. May (1868), as reproduced in M’Gowan [46] sible influence of environmental and metabolic factors’ suggested that these outbreaks were connected to some [56]. Given that prion strains constitute “a dynamic col- possible ‘immediate’ and ‘remote’ causes that arose from lection or ‘cloud’ of misfolded protein assemblies that adverse conditions and practices in the new and unfamil- are maintained under biological selection in a host and iar husbandry environment of merino sheep. its constituent tissues” [58], instances of atypical scrapie May’s thoughts in 1868 on the origin of classical may provide populations of prions susceptible to evolu- scrapie in eighteenth and nineteenth century France and tionary processes. Germany accord with the present hypothesis that prop- agating epidemics of classical scrapie can arise from sources in atypical scrapie. Three stages can be envisaged for the process of transformation from one manifestation 6 Discussion of scrapie to another and are shown in Figure 2. Stage one would concern the induction of atypical scrapie. Stage The hypothesis that atypical scrapie has an intrinsic two would concern the induction of classical scrapie from potential for acquiring the function of communicability atypical scrapie and stage three would concern the induc- and can be the progenitor of classical contagious scrapie tion of a propagating epidemic. Knowledge from epidemi- is supported by concepts from evolutionary biology and ology and pathology can illuminate the factors involved pathophysiology and by studies of the epidemiology, in these three stages and inform the development of bios- aetiology and natural history of prion diseases. Accounts ecurity measures and ways and means of optimising flock of strain behaviour and direct demonstration of natural health. selection in action show that prions in general and the An understanding of the determinants of atypical/ prions involved with scrapie possess the basic functions Nor98 scrapie and the first stage of a possible progression of variation, reproduction, and heritability [8, 59], which to a full-blown scrapie epidemic can come from epide- underpin evolvability [6]. The mechanism for these func- miology and pathology. Epidemiological studies define tions resides within the misfolded protein assemblies atypical/Nor98 scrapie as a non-communicable version of that constitute populations of disease-causing prions scrapie [17, 49, 57], which tends to occur in older animals [58] rather than within a genome based on RNA or DNA. and has a longer duration of illness than classical scrapie. As a consequence, transformation of atypical scrapie to In brief, epidemiological studies indicate that the founda- contagious classical scrapie by means of evolutionary tions of atypical/Nor98 lie within sheep and their environ- processes can be proposed as a conceivable event with ment and interactions between the two. intelligible causes related to the epidemiological trian- Proteostasis and the accumulation of misfolded gle of disease agent, host and environment [48]. Since protein that characterise prion diseases such as scrapie scrapie is the historical archetype of the prion diseases, are points of interest in the genesis of atypical scrapie and implications of prion evolvability go beyond the health of the situation can be explored with help from the concepts sheep and extend to all species affected by diseases that of allostasis and allostatic load [65-68]. These concepts impair processes for protein quality control [60-62] and unify notions of stress, homeostasis and physiological stability or eustasis, as opposed to cacostasis [69]. Allosta- 8 D. B. Adams

Figure 2: The three-stage process required for the endogenous emergence 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. sis refers to active processes that underlie adaptation to increases in misfolding may overwhelm the clearance intrinsic and extrinsic stressors and allostatic load refers mechanisms through proteasomes and by autophagy. to the cumulative effects of exposure to stressors and their Thirdly, clearance mechanisms or other components of adverse consequences in pathophysiology and dysfunc- the protein quality control system [62] may malfunction tion [69-79]. Allostatic load evokes the notion of wear and or any or all of the three possibilities may interact. Alter- tear and bears upon the second law of thermodynamics natively, there may be a general collapse in proteostasis and the need for continual repair and regeneration in occasioned by allostatic load. living organisms. Proteostasis or the quality controlled Reviews of the biological function of the cellular cellular processes for the production and turnover of pro- prion protein (PrPC) [71-73] do not hint at circumstances teins [61, 62] is central to repair and regeneration and thus that may increase rates of production of PrPC and heighten falls within the scope of allostasis. the risk of the misfolded conformer (PrPSc). However, a The functional failure that leads to atypical scrapie generalised causal link between neuroinflammation and may occur at three key places in the process of proteostasis signalled by the misfolding of protein as depicted earlier in Figure 1. Firstly, an induced demand [74] implies that virtually any infectious or non-infectious for PrPC may be accompanied by amounts of PrPSc that exposure that leads to neurological disease in sheep can exceed the adaptive capacity of protein clearance mech- be regarded as a potential risk factor for atypical scrapie anisms. Secondly, higher amounts of PrPSc resulting from and the list is extensive [75-77]. More specifically, certain Prion Evolvability and the Hazard of Atypical Scrapie in Small Ruminants 9 environmental exposures could increase rates of misfold- does not require mechanisms for portals of exit and entry ing of PrPC and lead to atypical scrapie given that swainso- of the disease agent and is demonstrated by experimental nine, an indolizidine alkaloid, was used to induce muta- inoculation and by instances where artificial exposures tions in prions and to demonstrate Darwinian evolution in caused common-source as opposed to propagating epi- prion strains [40, 79]. demics. Examples are bovine spongiform encephalopa- Swainsonine is a mycotoxin synthesized by endo- thy (BSE), where the large-scale transboundary epidemic phytic fungi and associated with poisonous plants such as was driven by feedstuffs containing products derived from Swainsona spp., Astralagus spp. and Oxytropis spp., which infected cattle, and iatrogenic Creutzfeldt-Jakob disease cause neurological diseases in sheep and other ruminants (iCJD) where outbreaks resulted from contaminated sur- [39]. Sheep are universally exposed to cyanotoxins, myco- gical instruments, contaminated extracts of pitu- toxins, phytotoxins and environmental toxicants and it itary glands and contaminated implants of corneas and is inconceivable that none would have consequences for dura mater [82]. If the circumstances of BSE and iCJD the misfolding of protein and similar implications for the were to recur with atypical scrapie in sheep, any ensuing onset of atypical scrapie. Cyanobacterial toxins associ- common-source outbreak would expand populations of ated with poor water quality present as a special concern scrapie prions available for evolutionary selection and because of their known connection to major neurodegen- favour the possible emergence of communicability and erative prionoid diseases in people [41-45]. Malfunctions classical scrapie. in protein clearance mechanisms that may trigger atypical Atypical and classical scrapie have differences in the scrapie are conceivable accompaniments to the inherited tissue distribution of misfolded prions (PrPSc) and infec- lysosomal disorders that occur in sheep [79]. tivity that may align with contagiousness. PrPSc and infec- Possible impacts of allostatic load on proteostasis and tivity are present as a constant feature in the lymphoretic- a consequential increased risk of atypical scrapie can be ular tissues and cells of sheep with classical scrapie. In viewed from a comparative medicine or ‘one health’ per- contrast, PrPSc is restricted to the central nervous system spective and with reference to the example of Alzheimer’s and not detected in the lymphoreticular tissues and cells disease in humans. Like scrapie, Alzheimer’s disease is a of sheep with atypical scrapie. This difference is cardi- proteopathy and a prion disease hinging on the seeded nal because the migratory behaviour of lymphocytes and aggregation and impaired clearance of disease-specific mononuclear phagocytes [29] provides a means for dis- proteins [64]. The incidence of Alzheimer’s disease in seminating the scrapie agent throughout the body and to white women in the USA from 1986 to 2006 was positively the multiple portals of exit that operate either prenatally associated with the social gradient; that is, women lower or post-natally. Exosomes may also be involved in the in the social or wealth spectrum had higher incidences of bodily distribution of the scrapie agent [83]. Significantly, the disease [81]. The social gradient builds on the social transmission studies with transgenic mice have detected determinants of health or the circumstances in which infectivity in lymphoid tissues, nerves and muscle of people are born, grow up, live, work and age, and the sheep with atypical scrapie occurring naturally or induced systems put in place to deal with illness [78]. In doing so, by intracerebral inoculation [23]. Accordingly, atypical the social gradient reflects chronic stress or allostatic load. scrapie is associated with a small population of prion A similar impact of chronic stress or allostatic load applies variants with an affinity for lymphoreticular cells and a to scrapie in sheep where a lower frequency of the disease propensity for communicability that could be expanded was associated with ‘less harsh farming conditions’ [56]. by evolutionary processes. Stage two in the notional progression from atypi- Current knowledge allows a broad appreciation of cal scrapie to classical scrapie and then to a full-blown where and when scrapie prions could move from the epidemic of scrapie concerns the acquisition of commu- central nervous system, what influences the presence of nicability in populations of prions generated in atypical prions in lymphoreticular cells and how atypical scrapie scrapie. Investigations of atypical scrapie have detected could transform to classical scrapie. The scrapie agent no animal-to-animal transmission of the disease and lack could move from the central nervous system through cer- of communicability can be deemed as the defining feature ebrospinal fluid (CSF) or interstitial fluid and in exosomes of atypical scrapie. The distinction between communica- or cells, particularly immune cells, which are known to bility and transmissibility has fundamental importance. traffic through nervous tissue as part of immune surveil- Contagiousness goes beyond transmissibility and entails lance [84-86]. CSF drains from the central nervous system inbuilt mechanisms for exit of the causative agent from through a network of lymphatic vessels in the dura mater one animal and entry to another. Simple transmissibility and then to cervical lymph nodes [87-88] and intersti- 10 D. B. Adams tial fluid drains to lymph nodes by means of pathways preventive measures can build from earlier recommen- within the walls of cerebral capillaries and arteries [84, dations [118] and emphasise breeding only with sheep in 89]. Exchange between interstitial fluid and CSF occurs robust health and the avoidance of lambing in confined through the glymphatic system, which comprises con- spaces where contact with contaminated ejecta and excre- nected periarterial and perivenous spaces [85, 86, 89]. tions may be unavoidable. In this regard, infectious prions Neuroinflammation may result in increased trafficking can persist in the environment [119] and particular soil of immune cells through the central nervous system [90] types for extended periods [120]. and an increased opportunity for prions associated with Given that the scrapie agent and prions in general atypical scrapie to spread from the brain. Thus, virtually are objects of selection and can evolve, the endogenous all infectious or non-infectious exposures that lead to generation of propagating epidemics of scrapie disease is neurological disease in sheep [75-77] can be reiterated as conceivable when particular conditions and risk factors potential risk factors for the transformation of atypical to coincide in the three-stage process for transformation classical scrapie. depicted in Figure 2. As a result, probabilities will be Another set of potential risk factors for the transfor- multiplicative, implying that the overall probability of mation of atypical to classical scrapie relates to increased endogenous epidemics of classical contagious will be affinity for lymphoreticular cells in prion populations. minuscule. However, the possibility of endogenous epi- Once again the field of candidate risk factors is large demics constitutes a clear-cut concern for veterinary ser- because the possibilities include conditions accompanied vices everywhere and by reason of the ecological fallacy by or perturbations to the lymphoreticular or fallacy of division, This informal fallacy refers to the system, or both. Precedents come from observations in ‘erroneous transference of an attribute from a whole (or sheep co-affected with scrapie and chronic inflammatory a class) onto its parts (or members)’ [121] and applies to conditions [91] and sheep with lentiviral infections [92-94]. epidemiology, where errors will arise when the aggre- The third stage in a possible transformation of atypi- gated behaviour of global populations is extrapolated to cal scrapie to classical scrapie is a propagating epidemic sub-populations without regard to circumstances [48, driven by scrapie prions with the function of communi- 122]. The model for veterinary services promulgated by cability (see Figure 2). Risk factors or component causes the World Organisation for Animal Health [14] proposes for this stage spring from transmission, which is known to ongoing capacity-building based on the disciplines that occur both prenatally and post-natally. Prenatal transmis- support comparative medicine. These disciplines include sion is firmly established and can result from the scrapie evolutionary biology [2, 123], which can assist in under- agent in semen or in the dam, and the conceptus can be standing important aspects of atypical scrapie in sheep infected at any time from oogenesis onwards [30, 95-98]. and goats and managing the possible consequences of Post-natal transmission can occur through environmental this disease. exposure to the scrapie agent derived from ovine excre- tions and ejecta such as faeces [99, 100], urine [101, 102], and foetal membranes [103-111], and in ovine secretions such as milk [92, 94, 112-114], oral secretions [115-117], and 7 Conclusions semen [97]. The component causes of atypical scrapie and its possible Infectivity, expressed mathematically as the propor- progression to classical scrapie are very broad and include tion of exposures to a disease agent that results in infec- virtually anything that impinges on the physiological sta- tion, was analysed in the records of six scrapie infected bility or allostasis of sheep and which may harm prote- flocks (2,060 offspring and 606 cases of scrapie) to gauge ostasis or favour the selection of prion variants with the the relative impact of prenatal and post-natal transmis- property of communicability. For this reason, actions that sion pathways [30]. Transmission from sire to concep- prevent atypical scrapie will fortify efforts to enhance the tus recorded an infectivity of 43% and transmission from health, welfare and productivity of sheep and will assist dam to conceptus plus transmission from dam to neonate towards the thermodynamic efficiency required for sus- had an infectivity of 44%. These prenatal infectivities tainable regenerative agriculture [124]. There are grounds, outweigh the 12% infectivity associated with post-natal however, for paying particular attention to water quality transmission of scrapie agent from the environment. They and exposure to cyanobacterial toxins, which have a signal that attention to risk factors associated with breed- known association with prionoid neurodegenerative dis- ing can block the possibility of a propagating epidemic eases in people [41-45]. arising from a rare occurrence of atypical scrapie. Broad Prion Evolvability and the Hazard of Atypical Scrapie in Small Ruminants 11

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