GENERAL INTRODUCTION: PRION DISEASES

Introduction

Scrapie is the oldest known transmissible spongiform encephalopathy (TSE) and was first described in sheep in 1750 in a German book on agriculture although others implied that it has been known since Roman times.¹⁶⁹ Many theories existed about the cause of the disease; hereditary, sexually transmitted and/ or infectious but only in the 20^th century was it demonstrated that the scrapie agent was transmissible.

Major events leading to our current knowledge of scrapie occurred as follows:

• 1898: Description of the microscopic neuropathology and neuronal vacuolation notably of motor neurons in the spinal cord.¹⁷
• 1936: Scrapie shown to be an experimentally transmissible disease in sheep.³⁴
• 1936 to 1938: Iatrogenic scrapie occurred in hundreds of sheep in Great Britain through the use of louping-ill vaccine contaminated accidentally with scrapie agent from brains and spleens of sheep used for its manufacture. This was first reported at the Third International Congress for Microbiology⁷⁵ and subsequently published in 1946.⁷⁴ The transmissible nature of the scrapie agent was thus confirmed beyond reasonable doubt.
• 1957 to 1959: Zigas and Gajdusek reported the occurrence of kuru (trembling or shivering of the body, also known as the laughing death), a rare and unusual neurological disease in the Fore-speaking people of Papua New Guinea.²²¹ They believed this was propagated by endocannibalistic consumption of the whole bodies of dead relatives during funeral rites. The American veterinary pathologist W.J. Hadlow made the important pathological connection between scrapie of sheep and kuru of humans by observing that ‘the overall resemblance between kuru and scrapie is too impressive to be ignored’.⁸⁰ He went on to suggest the experimental transmission of kuru to a laboratory primate. This was subsequently achieved and was followed by successful transmission of human Creutzfeldt-Jakob disease (CJD) to primates, thus establishing that these rare human disorders were, like scrapie, transmissible spongiform encephalopathies (TSEs).
• 1961: Experimental transmission of scrapie to mice thus establishing a model system in which the disease, and the agent that caused it, could be studied in the laboratory²⁶
• 1966 to 1967: Report of the very small size of the scrapie agent, its almost complete resistance to destruction by massive germicidal doses of ionizing radiation supporting the view that the agent may have a mode of replication independent of the integrity of a nucleic acid moiety.^{4, 5}
• 1979: Report of the scrapie replication site hypothesis⁴⁵ and the results of studies on the genetics of scrapie in sheep and mice.⁴⁴ The latter report showed that murine genes influenced the phenotype of scrapie, notably its incubation period and lesion profile, when a small number of important factors were controlled. This work was the basis for identifying and distinguishing strains of scrapie agent and led to further opportunities to study the pathogenesis of scrapie notably by Bruce, Dickinson, Fraser and Kimberlin. The scrapie strain typing methods were to become vitally important after the discovery of bovine spongiform encephalopathy (see Bovine spongiform encephalopathy).
• 1979, 1980 and 1982: Reports on the tissue distribution of scrapie infectivity in goats and Suffolk sheep and in some other breeds of sheep with natural scrapie, including during the incubation period of Suffolk sheep from high scrapie-risk families^82-84
• 1982: Establishment of the prion definition (small, proteinaceous infectious particles that resist inactivation by procedures that modify nucleic acids) and prion hypothesis.¹⁵⁷
• 1990: Description of prion protein polymorphisms at position 171 associated with incubation control of scrapie in sheep.⁶² Additional polymorphisms were later identified at positions 136 and 154; all associated with scrapie susceptibility in sheep. This formed the basis for scrapie eradication by genotype selection in various countries.
• 2003: Report of a new type of scrapie in Norway, Nor98,¹⁶ which was subsequently found in other countries, including those considered free from scrapie. This disease was later termed ‘atypical scrapie’ to distinguish it from ‘classical scrapie’ that has been known for centuries. Retrospective studies confirmed atypical scrapie in archived sheep material from the 1970s and 1980s,^{27, 213} which suggests that it is not a new disease.

Substantial epidemiological studies have failed to reveal a connection between CJD in humans and scrapie; these were summarized by the European Food Safety Authority in 2011.⁴⁶ Subsequently, it has been shown that some classical scrapie isolates can be transmitted to transgenic mice carrying the human prion protein gene and reproduce characteristics of a disease on second passage with similarities to sporadic CJD.²⁵ Whilst this may indicate that some isolates of the scrapie agent may be zoonotic, it is not known whether transmission is possible in natural circumstances.⁴⁹

Classical scrapie isolates have produced disease in cattle following intracerebral inoculation,^{29, 36, 115, 160} which was different to bovine spongiform encephalopathy, but there has been no evidence of infection following oral challenge.^{35, 119} Studies in the UK failed to infect pigs with classical scrapie,¹³⁵ which is contrary to more recent studies in the USA, that reported infection by intracerebral and oral routes.⁷⁷ Whether atypical scrapie can be transmitted to other farm animal species is currently unknown.

Classical scrapie has been found in most of the sheep-raising countries of the world at some time or another, often following importation of infected animals, but in some of these countries, including Australia, New Zealand and South Africa, the disease was eradicated before it could spread to indigenous sheep. The first case in South Africa was in 1966 in a Hampshire Down sheep imported from England.²⁰⁵ The disease was subsequently found in 11 sheep, all first- or second-generation progeny of the English imports of Hampshire Downs. A strict slaughter policy was applied, and no case has been diagnosed in South Africa since 1972, and the disease is considered to have been eradicated.

In contrast, atypical scrapie has been found even in sheep born in countries free from classical scrapie, such as New Zealand¹⁰⁶ and Australia.³¹

Aetiology

It is commonly accepted that scrapie is an infectious disease caused by prions, i.e. proteinaceous infectious particles that are devoid of nucleic acid.¹⁵⁸ The hypothesis is that the prion is composed exclusively of a modified isoform of PrP, named PrP^Sc. The latter is generated from the normal cellular form (PrP^C) by re-folding part of its α-helix coil structure into a β sheet. These physical changes to the tertiary structure of the protein profoundly alter its physical properties and, it is also claimed, confers strain-specific (biological) properties.² Thus, whereas PrP^C is soluble in non-denaturing detergents, the disease-specific form is not. PrP^C is denatured by proteases such as proteinase K but PrP^Sc is partially protease resistant, a feature that is utilized to aid in the diagnosis of prion diseases. PrP^Sc provokes no immune response in the host, presumably because the modified host protein, of which it is mostly comprised, is recognized as ‘self’. To prove the prion hypothesis, experiments usually relied on extraction of PrP^C from live animals, which may have also contained DNA from viruses or bacteria, but no nucleic acid has been found despite diligent searching. In fact, infectious prions have been generated de novo from bacterially-expressed recombinant PrP, which can cause disease in mice.²²⁰ Strains are characterized by their structural, biochemical and stability differences of the prion protein.²¹⁹

Prions are extraordinarily resistant to heat, ultraviolet and ionizing radiation, and most chemicals that inactivate microorganisms, although sodium hypochlorite and autoclaving in 2M sodium hydroxide are effective in destroying it.¹⁹⁰ The use of 20,000 ppm sodium hypochlorite for 1 hour is recommended for use in practice to decontaminate surfaces.¹⁰⁵

There are still questions about the function of the cellular prion protein because it is also found in various non-neural tissues. It may be involved in T cell, synaptic function or copper metabolism.²²² Animals devoid of prion protein, however, appear to be clinically healthy and viable as reported for Norwegian dairy goats that have a natural mutation in the prion protein gene.¹⁵ However, inflammatory responses may be aggravated in these goats, at least after experimentally induced pulmonary inflammation,¹⁶⁶ and it has been hypothesized that PrP may contribute to protection of vulnerable tissues against inflammatory damage.¹⁶⁵

Epidemiology

Sheep and goats are the natural hosts of scrapie although there are considerable differences in the epidemiology between scrapie in these two species. Whilst disease in goats has been often attributed to contact with scrapie-infected sheep, outbreaks also occur in the absence of sheep.⁹ Scrapie has also been described in moufflon, a species of wild mountain sheep occurring in Europe.²¹⁶

Classical and atypical scrapie occurs world-wide but the exact regional distribution is difficult to determine accurately due to the absence of a reliable ante-mortem test, differing scrapie test sensitivities, reliance on reporting of clinical suspects and the stigma attached to a declaration of scrapie occurrence. Mandatory sampling of at risk animals, such as fallen stock, often leads to discovery of cases, particularly those of atypical scrapie.⁵⁵ Stringently enforced import regulations have enabled island countries such as Australia and New Zealand to maintain what is generally regarded as disease-free status although atypical scrapie has been detected in these and other countries where classical scrapie has not been reported.

Susceptibility to scrapie, both classical and atypical, is determined by the prion protein gene of the animal. Researchers demonstrated that sheep experimentally infected with a pool of brains from classical scrapie-affected sheep (named sheep scrapie brain pool 1, SSBP/1) had different susceptibilities resulting in varying incubation periods. The incubation period was controlled by a single gene (Sip gene) with sA (short incubation) or pA (prolonged incubation) alleles.⁹⁶ It was subsequently shown that the Sip gene is identical to the PrP gene, which may vary between individual sheep and goats: multiple mutations (polymorphisms) exist, some of which are associated with differential susceptibility to scrapie, including disease phenotypes. Three codons in the sheep prion protein gene are important with regard to susceptibility: codon 136 (encoding arginine A or valine V), codon 154 (encoding arginine R or histidine H) and codon 171 (encoding glutamine Q or arginine R), usually written as VRQ/VRQ, ARQ/ARQ etc. Experimental studies in sheep infected with bovine spongiform encephalopathy, SSBP/1 and another isolate, CH1641 from a Cheviot sheep,⁵⁶ have shown that polymorphisms at codons 136 and 171 affected disease incidence and incubation time.⁶³ PrP alleles encoding for valine at codon 136 and glutamine at codon 171 (VRQ) are associated with increased susceptibility to classical scrapie, whereas those encoding for arginine at codon 171 (ARR) are associated with increased resistance.¹¹ These findings formed the basis for control programs to eradicate classical scrapie, such as the National Scrapie Plan in Great Britain (Table 1). Histidine at codon 154 also appears to have some protective effect against classical scrapie.⁵¹

Table 1 Classical scrapie risk categories used in the National Scrapie Plan in Great Britain

Although ARR/ARR sheep are the most resistant sheep, resistance is not absolute since there have been rare cases of classical scrapie in sheep of this genotype.⁷⁹ Other polymorphisms linked with resistance to classical scrapie have been found subsequently, such as K₁₇₆ (encoding lysine) or T₁₃₇ (encoding threonine).²⁰³

Susceptibility to atypical scrapie is also dictated by polymorphisms at these three codons but, contrary to classical scrapie, cases occur particularly in AA₁₃₆ sheep, even ARR/ARR sheep, and sheep carrying the AHQ allele, whilst being rare in sheep with the VRQ allele.¹⁴

Polymorphisms at codon 136, 154 and 171 are irrelevant for susceptibility to classical scrapie in goats, which are either ARQ/ARQ or AHQ/AHQ. However, other polymorphisms have been associated with increased resistance to classical scrapie in goats; the most important being at codons 222 (encoding lysine K), 146 (encoding serine S or aspartate D) and 211 (encoding glutamine Q)²⁰² (Table 2). Similar to sheep, H154 is associated with increased susceptibility to atypical scrapie in goats³⁰ although it may offer some protection against classical scrapie.^{18, 153}

Table 2 Classical scrapie risk in goats

M…Methionine, P…Proline, N…Asparagine, I…Isoleucine

Many studies have been carried out in various breeds to determine the frequency of alleles associated with resistance. These have shown that certain breeds may have little resistance in general or certain polymorphisms are breed- or region-specific. For example, Icelandic short-tailed sheep were all homozygous Q₁₇₁.¹⁹⁶ Whilst a high frequency of resistant genotypes (Q₂₁₁, S₁₄₆ and K₂₂₂) was detected in the goat population in Greece,¹⁰³ it was considerably lower in surveys in the United Kingdom (less than 7 per cent) and Boar goats were the main carriers of the S146 allele,^{64, 65} which was also the case for goats in Cyprus.¹⁵³

Increasing the prevalence of the ARR allele has been shown to reduce the overall risk of scrapie at the population level in sheep.¹⁵⁰ Indeed, genetic selection for scrapie resistance has led to a great reduction in the number of classical scrapie cases particularly in Europe, and especially in sheep where the influence of the prion protein gene on susceptibility has been studied the longest. The failure to effectively control scrapie in some affected European countries in the period between 2002 and 2012 was linked to regional variations in the implementation of genetic and non-genetic control measures. By contrast, the prevalence of atypical scrapie in Europe has been relatively consistent across countries, with approximately 6 cases per 10,000 tests. Testing for scrapie included fallen stock and healthy slaughter animals and was not just limited to neurological cases. Atypical scrapie is much rarer in goats, with an approximate prevalence of 1 per 10,000 tests, and only reported in five European countries.⁴⁸

Risk factors for the occurrence of classical scrapie other than genotype were found to be flock size (higher with larger flocks), soil drainage (higher with naturally wet soils), purchase of replacement sheep through a market, spread of sheep compost on the land and disposal of placentae in the compost.^{87, 188}

Scrapie is a disease of adult sheep and goats, usually between two and five years of age in classical scrapie⁴² although there are some reports of cases in sheep less than a year old.^{102, 172} Age-associated differences in scrapie incidence may be due to genotype, the level and age at exposure. Atypical scrapie generally affects older animals, above 5 years old.^{14, 30} Within-flock incidence can vary and is dependent on genotype distribution in the flock but can range from 1 to 20 per cent.⁴² In outbreaks of classical scrapie in sheep and goats, which led to herd cull with examination of central nervous and peripheral tissue for scrapie, a prevalence of above 30 per cent has been reported.^{33, 60}

Studies that determined presence of prion protein or infectivity in various organs, body tissues or fluids were vital to gaining a better understanding of the routes of transmission in scrapie. It is commonly accepted that the risk of horizontal transmission is greater in animals that have wide-spread accumulation of scrapie-associated PrP in the body, which is ultimately linked to prion protein genotype and scrapie strain.

In classical scrapie, transmission occurs both vertically and horizontally. Infectivity has been demonstrated in foetal tissues of a scrapie-infected sheep¹⁸³ and prion protein detected in foetal tissues by in vitro assay protein misfolding cyclic amplification (PMCA).⁵⁸ This is indicative of maternal, pre-natal transmission. Whether scrapie can be transmitted via semen or embryo transfer is still debated because studies have produced conflicting results, and interpretation is often difficult because of small sample sizes. Prions have been found in semen from scrapie-affected rams using PMCA and infectivity confirmed in one study¹⁶¹ whereas there was no evidence of infectivity in semen in another study.¹⁶⁷ Embryos collected from naturally-infected donors and implanted in scrapie-free sheep did not develop scrapie^{131, 212} but scrapie was confirmed in lambs produced by embryo transfer in experimentally infected recipient ewes.⁵⁷ It was concluded that the risk of transmitting scrapie via embryo transfer was negligible when using ARR homozygous or heterozygous embryos and adhering to OIE procedures relating to embryo transfer.⁴⁷ An epidemiological study found increased incidence of scrapie in the offspring of scrapie-affected dams but not in the offspring of scrapie-affected sires. That is suggestive of transmission from mother to offspring.⁹⁰ Such transmission may occur perinatally or postnatally in sheep and goats via contaminated placental tissues^{7, 168} or by ingesting colostrum and milk.^{114, 120} The latter may be more efficient in the presence of concomitant lentivirus infection of dams.^{122, 128}

Transmission of classical scrapie can also occur in the absence of contact with placentae or milk,¹⁶³ and many studies have now confirmed infectivity or presence of the responsible prion in a range of secretions and excretions, such as faeces,¹⁹² urine¹⁶² and saliva¹⁸⁹ and even skin,¹⁹⁵ which may all contaminate the environment.⁷⁶ This, together with the considerable resistance of prions to disinfection and biological inactivation, is the reason why scrapie is very difficult to eradicate once it has been established in a flock. It has been shown that the classical scrapie agent can remain biologically active in the environment for at least 16 years.⁶¹ Sheep on contaminated pasture can be infected in the absence of contact with infected sheep.⁴³ Furthermore, prions can be detected on objects such as fence posts and water troughs¹³³ and contact with such objects can result in  disease in sheep although weathering may reduce infectivity.¹¹³ Disinfection with sodium hypochlorite may also not prevent re-infection in contaminated environments.⁸⁵

Iatrogenic transmission of classical scrapie has also been reported in sheep vaccinated against Mycoplasma agalactiae.²⁴ There was no evidence that the use of pituitary-derived hormones used in sheep breeding were associated with an increased risk for development of atypical scrapie.¹³⁴

Little is known about transmission of atypical scrapie if it occurs at all. Results from an epidemiological study suggested that atypical scrapie was either not contagious or had a very low transmissibility under natural conditions.⁵³ No significant risk factors for transmission were found in atypical scrapie case control studies.^{40, 54, 92} Although multiple cases of atypical scrapie have been found in flocks and atypical and classical scrapie may co-exist in flocks, there is currently no epidemiological evidence that these two disease types are linked or caused by lateral transmission¹⁴⁹ and it may be a spontaneous disease.¹⁴ Experimental studies  confirmed that atypical scrapie can be transmitted via the oral route¹⁷⁷ and that the atypical scrapie agent can ‘shift’ into the classical scrapie strain CH1641 upon passage within sheep¹⁷⁸ but it is not known whether this can occur under natural conditions.

Pathogenesis

After natural or oral infection, the scrapie agent generally accumulates in tissues of the lymphoreticular system prior to neuroinvasion via ascending neural and hematogenous pathways.

The pathogenesis of classical scrapie has been studied in naturally infected, highly susceptible VRQ/VRQ sheep killed at sequential time-points:^{207, 208} PrP^Sc was detected in the gastrointestinal tract-associated lymphoid tissue of the palatine tonsil, the Peyer’s patches of the small intestine and retropharyngeal, caudal jejunal and ileocaecal lymph nodes at 2 months of age. Immuno-labelling in lymph nodes was restricted to tingible body macrophages. PrP^Sc immune-labelling was more widespread, involving other peripheral lymph nodes and spleen, at 3 months of age and also included follicular dendritic cells. PrP^Sc was found in the enteric nervous system from 5 months of age. Neuroinvasion was confirmed from 10 months of age: PrP^Sc was detected in coeliac and mesenteric ganglion complex, dorsal motor (parasympathetic) nucleus of the vagus nerve in the medulla oblongata and intermediolateral column of thoracic spinal cord segments T8-T10. At the end-stage (26 months of age), the entire enteric nervous system, brain and spinal cord was affected. The circumventricular organs that have a less effective blood-brain barrier, may also act as an entry point for prions to the central nervous system, i.e. via the haematogenous route since PrP^Sc has been found in these organs.¹⁷⁹

It has been shown that classical scrapie can be transmitted by blood transfusion, and infectivity in blood increased gradually during the incubation phase in VRQ/VRQ sheep.⁹⁴ Monocytes, lymphocytes and platelets were found to carry infectivity in blood, originating from lymphoid germinal centers and possibly bone marrow.¹²³

The risk of scrapie infection is correlated with the development of the Peyer’s patches and therefore highest in the first year of life. Thereafter susceptibility declines, presumably due to a reduction in surface area of ileal Peyer’s patches and lymphoid follicle density.¹⁸⁵

Differences in the time-course of neuroinvasion and peripheral involvement may occur depending on prion protein genotype or prion protein strain. In naturally infected ARQ/ARQ Suffolk sheep, PrP^Sc was first detected at 8 months in sequential tonsillar biopsies starting at 3 months of age.¹⁰⁰ No PrP^Sc immune-labelling was detected in ARR/VRQ sheep in a flock with natural scrapie up to 9 months of age whilst it was present in VRQ/VRQ sheep.⁶ PrP^Sc was found in a 3 month-old ARR/VRQ lamb in a different study.⁵² In some sheep or genotypes (ARQ/ARQ and ARQ/AHQ Sarda sheep) no evidence of peripheral PrP^Sc deposition was found, even though it was present in central and enteric nervous systems.¹²⁹

Oral infection of sheep with classical scrapie resulted in early and consistent PrP^Sc accumulation in peripheral lymphoid tissue in VRQ/VRQ, VRQ/ARQ and ARQ/ARQ sheep whereas it was minimal, inconsistent or occurred later in VRQ/ARR and ARQ/ARR sheep, and ARQ/ARR sheep had the longest survival time.⁷²

The finding of regular peripheral PrP^Sc accumulations in sheep with classical scrapie enables diagnosis in the live animal by examination of lymphoid tissue that is accessible thorough biopsies, such as third eyelid,¹⁴⁴ tonsil¹⁰⁰ and rectal mucosa.⁶⁷

Although the pathogenesis of classical scrapie in goats is fundamentally the same, less is known in relation to breed, prion protein genotype and age. This was studied in 200 selected goats from a classical scrapie outbreak which demonstrated that peripheral PrP^Sc accumulation was frequent (68 of 72 scrapie-affected goats) although it did not necessarily include lymphoid tissue of the rectal mucosa. Also goats homozygous for isoleucine at codon 142 (II₁₄₂) were more frequently infected than goats with a methonine allele (IM₁₄₂ or MM₁₄₂).⁷¹ Furthermore, the absence of detectable PrP^Sc in the enteric nervous system and thoracic spinal column, despite presence in the brain, suggested that the hematogenous route contributed significantly to neuroinvasion.⁷⁰ The consistent involvement of the coeliac and mesenteric ganglion complex in scrapie-affected goats in Cyprus suggested that the spread from the enteric to the nervous system is similar to sheep.¹⁴³

The pathogenesis of atypical scrapie is largely unknown, which is complicated by the majority of cases being detected by active surveillance of fallen stock or healthy slaughter animals and tissues for further examination are limited (brain only) or unsuitable (autolysed). Unlike classical scrapie, PrP^Sc is not found in peripheral lymphoid tissue.¹⁷⁵ This suggests that the agent is predominantly restricted to the central nervous system.

Transmission studies in transgenic mice carrying the ovine prion protein gene (tg338 mice), however, demonstrated infectivity – although not always consistently – in peripheral tissues from field cases, such as retropharyngeal, popliteal, pre-scapular and parotid lymph nodes, but it was impossible to determine whether this was due to oral exposure similar to classical scrapie or due to centripetal spread from the brain.⁸

Oral challenge of sheep within 24 hours after birth resulted in atypical scrapie with infectivity demonstrated in the distal ileum at 24 months of age,¹⁷⁷ i.e. considerably younger than that seen in natural cases of atypical scrapie.

It is currently not known which experimental infection route best mimics natural disease but the intracerebral route may be more suitable for a disease that presumably originates in the brain. AHQ/AHQ sheep inoculated intra-cerebrally with atypical scrapie brain homogenate presented with a uniform disease phenotype and had survival times between 378 and 1057 days, the mean of which was approximately half of the average age at death in field cases.¹⁷⁶

Clinical signs

Both classical and atypical scrapie are slowly progressive neurological diseases. Although PrP^Sc is found throughout the brain in clinically affected animals, the sometimes poor correlation between clinical disease or clinical signs in particular, and detectable PrP^Sc raises questions about the cause of clinical signs.¹¹¹

A detailed description of the clinical presentation of classical scrapie in sheep is given by Parry,¹⁵⁴ although that was written prior to the discovery of atypical scrapie and the use of more sensitive post-mortem tests.

Most cases of scrapie have an insidious onset and progress slowly over several weeks but progression over several months has also been observed.^{16, 154} Usually, only one or a few animals are affected simultaneously in a flock or herd. Early clinical signs may not be noticed, especially in flocks or herds that are not closely monitored. It is questionable whether acute or peracute scrapie cases occur that do not display premonitory signs of illness. In one survey, 16 per cent of confirmed classical scrapie cases were found dead without exhibiting signs before death.²⁸ It is more likely that clinical signs are not recognised or clinical signs are too subtle to be associated with scrapie. That would explain why so many cases, particular atypical cases, are detected by active surveillance in supposedly clinically healthy animals or fallen stock.

Early clinical signs can be fairly non-specific and are thus most likely to be observed by someone familiar with the animal. Farmers may suspect early scrapie in sheep or goats before neurological signs are evident due to their experience with the disease. These early clinical signs include weight loss and subtle behavioural changes, such as separation from other animals or changed behaviour during milking.

The main difference in the clinical presentation between classical and atypical scrapie is pruritus, which is largely absent in atypical scrapie. However, not all classical scrapie cases present with pruritus. Nevertheless, if pruritus is present it is usually easily spotted, especially in more advanced cases due to the loss of wool or hair as a result of frequent rubbing, nibbling and scratching. Circling has been described in cases of atypical scrapie, particularly in experimentally-induced disease.^{173, 175-177} This sign is rarely observed in sheep with classical scrapie although it has been reported in goats.¹⁰⁹ In general, detailed descriptions of clinical signs of atypical scrapie are rare because very few natural cases are identified as clinical suspects.

Clinical signs can be crudely grouped into three different categories, viz. changes in mental status, behaviour and activity; changes in sensory responses and changes in movement.

Changes in mental status, behaviour and activity

These are often the earliest clinical signs noticed. There may be separation from the rest of the flock or herd and the animal may become more prone to flight when approached and thus difficult to catch. Classical scrapie-affected animals, particularly goats, may also appear hyper-alert, may become more excitable and display exaggerated responses to external stimuli, such as sudden movements or noises. Repeated startle responses to hand clapping were seen in approximately 30 per cent of 129 sheep with classical scrapie in Ireland.⁸⁸ When left undisturbed, scrapie cases may appear somnolent or dull with a vacant gaze and low head carriage but this is more obvious in more advanced scrapie cases.

Sheep with classical scrapie tend to spend more time lying down than healthy sheep, are usually more inactive during these periods and are less socially responsive.⁸⁶ Teeth grinding, either spontaneous or during handling, is frequent in sheep with classical scrapie. Appetite is usually not affected until the very late stage of the disease. Abnormal drinking patterns have also been reported in the early stages of classical scrapie; affected  animals may drink more frequently but only small amounts taken.¹⁵⁴ Difficult milking is a common sign in sheep and goats with classical scrapie.²³

Changes in sensory responses

Classical scrapie is usually associated with increased grooming behaviour; affected animals spend more time rubbing on objects (e.g. fences, troughs), scratching (e.g. with horns), and nibbling themselves. The latter response is particularly frequent in goats.¹¹¹ In the later stages, rubbing may be accompanied by lip-licking and smacking (‘spontaneous nibble reflex’). These responses to pruritus are, however, often only displayed when the animal is undisturbed or accustomed to an observing person. The response to pruritus is utilized as clinical test, i.e. scratching of the back often results in lip-licking and nibbling (termed ‘nibble reflex’ even though it is not a reflex (Figure 1)) but some animals may merely respond with head or body movements, which is why the term ‘positive scratch test’ may be more appropriate. Pruritus eventually leads to wool and hair loss, usually on the poll, around the tail base, lateral thorax and lower lateral neck in sheep^{111, 154} (Figures 2 and 3). Further consequences of intense rubbing may be discolouration, hyperpigmentation and lesions of the skin. Skin lesions initially present as superficial abrasions, excoriation with crusts if allowed to heal and lichenification. Secondary infections with skin pathogens, such as Staphylococus aureus, Dermatophilus congolensis and parapox virus (orf) may also occur.¹⁰⁸

Figure 1 Positive scratch test in a goat with classical scrapie. Scratching of the back elicits head movements and lip licking

Figure 2 Three classical-scrapie-affected sheep but only one had obvious wool loss on the back

Although pruritus is generally associated with classical scrapie, it may not be present in all cases; this non-pruritic form was termed ‘ataxia type’, ‘paralytic form’ or ‘nervous form’^{102, 111, 148, 156} of scrapie. This is obviously dependent on the level of observation.

In a study of 162 classical scrapie-affected sheep, five (3 per cent) did not appear to be pruritic based on the absence of wool loss, scratch response and pruritic behaviour during 2.5 hours of passive observations.¹⁰⁸ Such differences may be due to prion protein genotype¹¹⁸ and strain, such as atypical scrapie or CH1641 which usually do not cause pruritus in sheep.^{14, 178}

There are infrequent reports of scrapie cases running into objects, possibly due to visual impairment.^{199, 204} This has been also described in atypical scrapie in both sheep and goats.^{141, 173}

Easier to evaluate is the menace response, which may be absent in one or both eyes. This was observed for classical scrapie in approximately 10 per cent of sheep in a study in Ireland⁸⁸ and 2 per cent of goats in a study in England.¹¹¹ It was also reported for sheep with atypical scrapie,^{112, 176} (Figure 4) and may be a feature of cerebellar disease.

Figure 3 A goat with classical scrapie and diffuse hair loss, which is more evident and accompanied by skin lesions on poll, upper neck and shoulder region

Figure 4 Absent menace response in a sheep with atypical scrapie. The sheep does not blink when the hand is moved towards the eye

Changes in movement

Head tremor was seen in over 40 per cent of sheep^{88, 108, 209} and in 7-10 per cent of goats with classical scrapie.^{111, 218} It has also been described in sheep with atypical scrapie.^{112, 176} Frequency of occurrence increases as the disease progresses.

Gait abnormalities found in both scrapie types include incoordination, hypermetria (high stepping gait), jumping of the hind limbs (bunny hopping), a stilted or hypometric gait, lack of balance, falling and difficulty in rising, eventually resulting in permanent recumbency and death.^{16, 88, 111, 112, 141, 200, 210, 218} Collapsing episodes, which may be a form of cataplexy-narcolepsy and may be triggered by handling stress or when chased, have been reported in sheep and goats with classical scrapie.^{114, 121, 155, 170} Postural changes may be present in combination with gait abnormalities, such as a wide-based stance or crouching.^{88, 111}

Circling, either spontaneous or when blindfolded, has been observed in sheep with atypical scrapie in both natural^{147, 173} and experimental disease.^{175, 176} It may also be seen occasionally in goats with classical scrapie.^{81, 109, 125}

Other clinical signs

Hypersalivation, or more likely excess saliva due to swallowing deficits, which may increase the risk of aspiration pneumonia, may be observed in sheep and goats with classical scrapie.^{9, 23, 89, 127}

Weight loss or loss of body condition is a frequent but non-specific sign, which has been described for all scrapie types in sheep and goats. There are no obvious physical changes (temperature, heart rate, respiratory rate) in scrapie cases.

There are no significant alterations in blood or cerebrospinal fluid that would be useful for ante-mortem diagnosis.

In the absence of a reliable live animal test, the clinical examination is currently the only method for diagnosis in live animals. However, as none of the clinical signs are pathognomonic, not all scrapie cases present with the full range of clinical signs and the clinical picture is affected by scrapie strains, confirmative tests following post-mortem are always necessary. Few protocols have been published to aid in the clinical diagnosis of classical scrapie in sheep^{37, 210} or scrapie in general¹¹⁶ based on the most frequent signs associated with the disease.

Other electrodiagnostic tests have been studied for use in sheep with scrapie, such as electroretinograms^{159, 180} and auditory evoked potentials,¹¹⁷ and although abnormalities were detected, these tests are expensive, time-consuming and have limited practical value.

Classical scrapie in goats

Atypical scrapie in sheep

Classical scrapie in sheep

Pathology

There are no pathognomonic gross lesions associated with scrapie. Morphological microscopic lesions are restricted to the central nervous system (CNS). The consistent microscopic lesions are vacuolar changes in the grey matter neuropil or neuronal perikarya, which are usually bilateral symmetrical, accompanied by astrocytosis.^{181, 217} Other forms of neuronal degeneration, such as chromatolysis or neuronophagia, may also be seen, and in some cases perivascular amyloidiosis may be present.^{215, 217} The latter appear to be more frequent in sheep with a V136 allele.¹³⁰

Intraneuronal vacuolation is not a feature in atypical scrapie cases¹⁴⁰ but descriptions of histopathological changes in atypical scrapie are limited because the brain material from confirmed cases is often autolysed.

Pathological changes have been observed in the retina, characterized by atrophy and loss of nuclei in nuclear layers and Müller glia hypertrophy in sheep with classical scrapie.⁹³

Diagnosis

The standard method is the microscopic examination of the brain. Histological examination should be performed on formalin-fixed, paraffin embedded brain sections that are stained with haematoxylin and eosin (H&E). The histopathological changes in the CNS, predominantly vacuolation, are visible in the target areas at the level of the obex for classical scrapie, i.e. the dorsal motor (parasympathetic) nucleus of the vagus nerve (DMNV) (Figure 5), and the spinal tract nucleus of the trigeminal nerve (STNT) (Figure 6). In atypical scrapie, vacuolar changes are less prominent in the brainstem whereas neuropil vacuolation may be observed in the molecular layer of the cerebellum and the cerebral cortex.¹⁶

Figure 5 Intraneuronal vacuolation in dorsal vagus motor nerve nucleus of a sheep with classical scrapie. H&E, 100× magnification

Figure 6 Spongiosis in the spinal tract nucleus of trigeminal nerve in a sheep with classical scrapie. H&E, 20× magnification

Immuno-histochemical analysis (IHC) can also be performed on paraffin embedded brain tissues in order to highlight the presence of PrP^Sc accumulation. The samples are deparaffinated, rehydrated, pretreated with 98 per cent formic acid and autoclaved at 121°C, then incubated at 4°C with monoclonal primary antibody anti PrP^Sc, incubated with avidin-biotin-peroxydase, reacted with chromogen 3-3’ diaminobenzidine (DAB), and counter-stained with Mayer’s hemalum. Different immuno-histochemical types of PrP^Sc deposition (see Bovine spongiform encephalopathy) can be observed in the brain of scrapie affected animals, which can be used to distinguish different scrapie strains. In sheep with classical scrapie, PrP^Sc is most frequently detected in the DMNV and less frequently and intensely in other neuroanatomical sites of the brainstem, such as solitray tract nucleus, STNT, midline raphe, olivary nuclei and accessory cuneate nucleus.¹⁶⁴ PrP^Sc is frequently visible as granular or punctate deposit in the DMNV and STNT.¹⁸² Glial-type labelling with PrP^Sc is able to identify deposits branching out from the nucleus of glial cells on their processes and giving them a stellate appearance is predominantly found in the molecular layer of the cerebellum and granular pattern is frequently present in the granular layer of the cerebellum (Figure 7). A detailed description of the PrP^Sc types in sheep brains with scrapie has been published.⁶⁹

Figure 7 Glial pattern in the molecular layer and granular pattern in the granular layer of cerebellum in a sheep with classical scrapie. Immunohistochemistry, antibody F99/97.6.1 (mouse, isotype IgG; Lot.P120227-001; VMRD, Pullman, WA, USA; 1:1000 dilution), 20× magnification

Figure 8 Granular pattern in both molecular and granular layers of cerebellum in a sheep with atypical scrapie. Immunohistochemistry, antibody F99/97.6.1, 10× magnification

The distribution of PrP^Sc is more restricted in atypical scrapie in sheep and the severity usually reduced when compared with classical scrapie. Immunolabelling is consistently greater in cerebellum than in the brainstem, and if detected at the level of the obex, restricted to the STNT.^{14, 140} That is why examination of the cerebellum, in addition to the brainstem, is advisable in small ruminants. Immunolabelling types of PrP^Sc are less variable in brains from atypical scrapie cases compared to classical scrapie, and intracellular and stellate PrP^Sc deposits, which feature in classical scrapie, are not evident in atypical scrapie brains. Fine granular immunolabelling in both molecular and granular layers of the cerebellum (Figure 8), but not stellate-type labelling, are most frequently seen in atypical scrapie brains.¹⁴⁰ PrP^Sc was not detected in the cerebellum in a goat with atypical scrapie but the presence of immunolabelling in more rostral areas and only in the STNT in the brainstem¹⁷¹ is consistent with the findings in sheep with atypical scrapie. More data on goats with atypical scrapie is needed.

Another PrP-based confirmatory test is immunoblotting, which demands fresh or frozen tissue. It is also suitable for use on autolysed tissue⁹⁵ that cannot be fixed for immunohistochemical examination. After digestion with proteinase K and labelling with anti-PrP-antibodies, three immunostained bands, di-, mono- and unglycosylated, of proteinase resistant PrP^Sc (PrP^res) are distinguishable (Figure 9). The highest (diglycosylated) band of approximately 27 kDa and the lowest (unglycosylated) band of approximately 19 kDa occur in classical scrapie.¹⁸⁷ Banding patterns are different in atypical scrapie (Figure 10) where an additional lower band of around 11 kDa is detectable.^{14, 201}

Figure 9 Western Blotting analysis of positive and negative classical scrapie cases

C-: negative control; C+: classical scrapie control; Lanes S1, S3, and S4: classical scrapie cases; lane S2: negative case. Immunodetection was performed with monoclonal antibody P4. This image was kindly provided by Dr. Mazza Maria, IZS Turin.

Figure 10 Western Blotting analysis of classical and atypical scrapie cases

C-: negative control; C₁+: classical scrapie control; C₂+: atypical scrapie control (Nor 98); Lanes S1: negative case; Lanes S2: atypical scrapie case; Lane S3: classical scrapie case. Immunodetection was performed with monoclonal antibody P4. This image was kindly provided by Dr. Mazza Maria , IZS Turin.

In addition to comparing molecular mass and glycoform ratio using a single antibody, monoclonal antibodies targeted at different epitopes of the prion protein can also be used to visualize different PrP^res fragments in Western immunoblots. This enables differentiation of classical scrapie from bovine spongiform encephalopathy (BSE) in sheep or goats and CH1641 scrapie.^{97, 124, 187} The latter is a distinct classical scrapie strain in both sheep and goats with molecular properties similar to BSE, which is thus diagnostically more challenging.⁹¹ Similarly, strain differentiation is possible by immunohistochemical examination of fixed tissue using a panel of different monoclonal and polyclonal antibodies. These recognize specific PrP^Sc sequences in specific cell types of the brain and lymphoreticular system (“epitope mapping”), which result in immunolabelling patterns that are characteristic for BSE, classical scrapie and CH1641 scrapie.^{98, 99, 198}

In countries where animals are actively monitored for scrapie, for example by testing fallen stock or healthy slaughter animals, rapid (screening) tests are usually applied to brain samples and any positive or inconclusive sample is then subsequently examined by immunohistochemistry and/ or Western immunoblotting for disease confirmation as described in the OIE Manual of diagnostic tests and vaccines for terrestrial animals.¹⁴⁵ A range of screening tests is available, e.g. enzyme-linked immunosorbent assays (reviewed by⁵⁹), which either detect PrP^res or do not use any proteinase digestion at all, although their diagnostic sensitivity may not be equal, which affects the efficacy of active surveillance systems.¹⁹ These tests may be inferior to immunohistochemistry in their sensitivity, particularly in goats, where cases may be missed if only screening tests are used.^{32, 71, 139, 151}

As PrP^Sc is usually detectable in lymphoid tissue in most sheep and goats with classical scrapie and generally earlier than in brain tissue, this tissue type could also be examined for scrapie diagnosis although the rapid screening tests have not been formally evaluated for use on tissue other than brain. Accessible lymphoid tissues suitable for testing in live animals are the nictitating membrane, palatine tonsil and rectal tissue containing recto-anal mucosa-associated lymphoid tissue (RAMALT). Only the latter has been proposed as a live animal test^{66, 68} because it is less challenging, can be done under local anaesthesia of the rectum, usually yields adequate sample size, and is commercially available. Single-use specula exist to aid in the collection of these biopsies. Diagnostic sensitivity is obviously dependent on the peripheral distribution of PrP^Sc, which is influenced by strain (e.g. no detectable PrP^Sc in atypical scrapie), genotype (e.g. more limited peripheral distribution in sheep carrying an ARR allele) and species (less successful in goats with classical scrapie.⁷¹

The probability of detecting scrapie in small ruminant carcasses is highest if both brain and lymphoid tissue are tested because of PrP^Sc accumulation in peripheral tissue prior to neuroinvasion in most cases of classical scrapie. Limiting testing to brain by rapid tests may identify classical scrapie only in the last quarter of the incubation period.¹⁰ Testing retropharyngeal lymph node tissue using rapid tests has been considered for the national scrapie surveillance programme in New Zealand.^{107, 211} Other ultrasensitive PrP-based tests that exist make use of the ability for prions to convert PrP^C into PrP^Sc or amyloid fibrils, such as PMCA and Real-Time Quaking Induced Conversion (RT-QuIC).¹⁰⁴ These could potentially be used as a live animal test since PrP^Sc has been detected by PMCA in blood from classical scrapie-infected sheep.¹⁹⁷ However, these tests are currently only used in specialist research facilities and have not been tested on a large number of animals infected with the various scrapie strains that exist.

For surveillance purposes, it is usually sufficient to detect scrapie and to further classify it as atypical scrapie, classical scrapie or BSE. Some scrapie isolates, particularly CH1641, can produce equivocal results where BSE cannot be excluded, and these are usually further investigated by transmission to a standardized panel of transgenic mice. The gold standard for strain characterization of the agent is still the mouse bioassay, which is however lengthy, expensive and impractical on a large scale although the creation of transgenic mice carrying the ovine prion protein gene has resulted in greatly reduced incubation periods. Strains can be differentiated in mice by clinical signs,⁴¹ incubation period,²¹ transmissibility,^{194, 214} histopathological lesion profile,²² imunolabelling profile,¹³ resistance to chemical or physical inactivation,¹⁹¹ resistance to proteinase K digestion¹²⁶ and PrP^res immunoblot profile.¹⁹³ An overview of rodent models available for strain typing has been provided.⁷⁸ Additional transgenic mice strains may become available as research tools for prion disease research.

Multiple classical scrapie strains exist, as demonstrated by mouse bioassays, such as ME7, 221C²¹ or strains that transmit poorly to wild-type mice.¹⁹⁴ The current immunoblotting methods are unable to distinguish these strains, except for CH1641, but this may be possible by immunohistochemistry. Different immune-labelling patterns in the brain in classical scrapie-affected ARQ/ARQ sheep from different countries is suggestive of different scrapie strains⁷³ although these patterns may also be affected by PrP gene polymorphisms at codons 136, 154 and 171.¹⁸² Vacuolar lesion profiles were found to be less useful to differentiate scrapie strains due to their high individual variability.^{12, 73}

Differential diagnosis

Scrapie is a slowly progressive neurological disease, which should rule out diseases with an acute onset and short clinical course, such as hypomagnesaemia, botulism, rabies and pseudorabies (Aujezsky’s disease) although the level of prior observation is crucial in this respect. Infectious neurological diseases, such as Aujeszky’s disease or listeriosis, are usually associated with fever or other distinct clinical signs (facial paralysis in listeriosis) although the latter has been confused with classical scrapie in goats.¹⁰⁹ Additional diagnostic tests, such as serology or blood biochemistry can be used to rule out other diseases (hypomagnesaemia, maedi-visna, caprine arthritis-encephalitis, ketosis) or there could be a response to treatment (polioencephalomalacia/ cerebrocortical necrosis/ thiamine deficiency). Abscesses, tumours or other space-occupying processes in the brain (e.g. cysts caused by larvae of the tapeworm Taenia multiceps) may cause clinical signs similar to atypical scrapie (circling) depending on the location of the lesion but are often also associated with extreme dullness, which is unusual for scrapie unless at the terminal stage. An overview of neurological diseases in sheep and goats and a list of diseases confused with scrapie is given by Brewer²⁰ and Scott and Henshaw¹⁷⁰ respectively. In a Swiss study of small ruminants with neurological signs, a goat with a brain tumour and ectoparasites had a clinical picture similar to scrapie.¹³⁶

Pruritus can be persisting and caused by ectoparasites. Pruritus caused by Psoroptes ovis can be diagnosed by careful clinical examination coupled with parasitological examination of appropriate skin samples. Pruritic scrapie cases usually lack the skin lesions (crusts, serum-oozing papules) seen in sheep with mange. Emaciation may be caused by malnutrition (nutritional, bacterial or parasitic causes), which may also affect fleece quality, or dental disease but should not cause any neurological signs.

A range of plant poisonings can cause syndromes characterized by gait abnormalities and tremor¹⁴² but these affect usually groups of animals, have an acute onset and the clinical history may allow differentiation from scrapie.

Immunohistochemically, it has been shown that increased PrP immunolabelling in the brain may occur in a range of neurological conditions of sheep; such occurrence needs to be distinguished from immunolabelling in scrapie cases. Labelling of neurons has been associated with axonal swelling, neuronal necrosis and lysosomal storage disorders whilst labelling in glial cells was associated with bacterial infections and linked to possible oxidative stress.¹⁰¹ These studies support previous suggestions that altered PrP expression or transcription is associated with oxidative stress.

Control

As scrapie is a fatal disease and successful treatment does not exist, disease prevention is the only long-term control option once the diagnosis has been established.

Passive surveillance, which relies on reporting and examination of clinical suspects, is often the only method in many countries to monitor the disease but is less likely to detect disease particularly if present at low prevalence. Clinical suspects diagnosed as atypical scrapie are extremely rare, even in countries where the disease occurs, and targeted surveillance for scrapie by testing fallen stock and healthy slaughter animals is more likely to identify the disease.¹³⁷

To determine the classical scrapie status of a country, zone, compartment or establishment certain criteria are necessary according to the World Organisation for Animal Health (OIE) Terrestrial Animal Health Code:¹⁴⁶

• A risk assessment identifying all potential factors for scrapie occurrence (e.g. feeding practices, import of feedstuffs, sheep and goats, semen and embryos) and their historic perspective;
• an awareness programme to enable recognition of the clinical signs and
• a surveillance and monitoring system.

Several conditions have to be met before scrapie freedom can be considered, which includes testing of animals for scrapie and taking appropriate measures with regards to feeding practices and imports of animals to prevent introduction of disease, which are outlined in articles 14.8.2 – 14.8.5 of the chapter.

Scrapie prevention in countries with endemic classical scrapie is primarily achieved by genotype selection, i.e. removal of sheep with a susceptible prion protein genotype (VRQ) and increasing the number of sheep with a resistant genotype (ARR) will reduce susceptibility to classical scrapie in sheep. Such programmes are in place in many countries. A breeding program was set up within the European Union in 2003 to select for resistant genotypes¹³⁸ although this may ultimately increase the number of animals in the population that are susceptible to atypical scrapie. However, detection of atypical scrapie does not usually result in the same strict legislative measures as for classical scrapie. In the European Union, any holding (establishment) where a confirmed case of atypical scrapie has been identified is currently subject to surveillance for two years. That involves testing of healthy slaughter animals and fallen stock over the age of 18 months.⁴⁸

Studies in sheep have not shown any unfavorable association between the ARR allele and milk performance and certain production and performance traits.^{3, 38, 39}

Reliance on tests following post-mortem alone to identify scrapie-infected animals without control involving genotype selection is unlikely to eradicate the disease.³² A similar genotype-based approach has not yet been tested in goats although it should be equally effective. The lack of resistant genotypes in certain goat and sheep breeds in some countries may present a problem, and herd cull has been used as an alternative option to eliminate the disease. However, re-introduction of the disease is likely if restocking is carried out using animals with susceptible genotypes given the persistence of the scrapie agent in the environment. In Iceland where a genotype-based approach is impossible due to the lack of sheep with resistant genotypes, current control measures include herd cull, destruction of hay, cleaning and disinfection of sheep barns and equipment by high pressure washing with soap and hypochlorite solution, replacing all worn materials in the sheep barn and/ or replacement with new buildings, restocking after a minimum of 2 years with lambs from scrapie-free areas remote from affected quarantine zones and continuous health control of new flocks to detect possible recurrence.^{61, 132} This has, however, not prevented re-occurrence. Mass restocking after whole herd cull increases the likelihood of re-occurrence of classical scrapie in goats.¹⁵²

Scrapie eradication has become more important with the discovery of bovine spongiform encephalopathy (BSE) in cattle because the latter is zoonotic and its link to feeding of contaminated meat and bone meal to which small ruminants may have also be exposed. Whilst there has been no evidence of naturally occurring BSE in sheep so far,¹⁸⁶ it was diagnosed in goats in the United Kingdom¹⁸⁴ and France.⁵⁰ The pathogenesis of BSE in goats is similar to classical scrapie in sheep²⁰⁶ and goats.¹ The BSE agent can also be transmitted to sheep via blood.⁹⁴ This is important in terms of risk for humans, should the BSE agent be confirmed in sheep under field conditions. Sheep were also used as a model to study transmission of variant CJD disease by blood products in humans because of similarities between the pathogenesis of TSEs in sheep and variant CJD in humans. Clinically, BSE is indistinguishable from classical scrapie in sheep¹¹⁰ and goats¹¹¹ based on experimental studies, although BSE-affected sheep generally present with pruritus. BSE can be differentiated from scrapie by the diagnostic tests mentioned above although classical scrapie-BSE co-infection, if it occurs, may be more difficult to identify and may require mouse bioassay or other tests, like PMCA.¹⁷⁴

The exclusion of known infectious materials (specified risk materials) from the food chain in the European Union is another measure to minimise exposure of humans to TSEs from any ruminant.

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