2 THE DISEASE
2.1 General considerations
Scrapie is a relentlessly, progressive, neurodegenerative, afebrile disease of adult sheep characterised by clear, but not unequivocal signs affecting behaviour, posture and movement, sensation, mental status and general condition. The clinical onset is usually insidious and the period of signs can be quite short (days or weeks). It seldom lasts for many months, especially as intervention by the farmer frequently secures an early death on welfare grounds or because such an animal interferes with flock management. Some cases are reported to be found dead without premonitory signs (Clark, Moar and Nicolson, 1994). In some countries the occurrence of the disease may be concealed by the pedigree breeder because knowledge of its existence may affect sales of breeding sheep. Even in countries where the disease is notifiable reporting may be discouraged unless the benefits of reporting (e.g. payment of compensation) outweigh the detriment. Previous experience of the disease hastens the farmer to take action to remove an affected animal at an early stage as there is no cure and, if the sheep is heavily pregnant, there is a potential danger to other sheep from the placenta (Pattison et al., 1972, 1974) and other discharges which may carry the scrapie agent. For further information see Kimberlin (1981) and Detwiler (1992).
2.2 The clinical disease
The clinical signs include pruritus (without scab formation as occurs in mange) but this, as with other signs, is not invariably present. Other signs are gait ataxia, trembling (hence la tremblante in French), trotting (hence traberkrankheit in German), loss of bodily condition, teeth grinding, apprehension and the presence of a nibbling reflex when the back is scratched.
2.3 The pathology and diagnosis
The pathological findings are confined to the brain and spinal cord and are microscopic. They include spongiform change in grey mater neuropil, vacuolation of neuronal cell bodies in specific sites, astrogliosis and neuronal cell loss. Sometimes amyloid deposits are found. These stain positively with conventional stains for amyloid and for PrP (see Paragraph 2.12 below). Such PrP staining can also be found associated with spongiform change. PrP, without accompanying morphological change, occurs also in other tissues, notably those of the lymphoreticular system of affected sheep and goats (e.g. in lymph nodes, spleen, tonsil and lymphoid tissue of the gut and third eyelid) including during the incubation period. PrP, can also be detected by immunoblotting of unfixed tissues. The pattern of morphological change in the brain of affected sheep, varies between outbreaks and breeds probably reflecting differences in agent strain and host genotype (Wood et al., 1997). Scrapie-associated fibrils (SAF), comprised of an aggregated form of PrP or a shortened form of the molecule can also be detected in detergent extracts of CNS treated with proteinase K and electron-densely stained for examination by electronmicroscopy (Hope et al., 1988; Scott et al., 1990) Confirmation of a clinical diagnosis is therefore made by one or other of the following methods:
New methods of diagnosis are being developed but most are variations upon existing methods or using different tissue sources. Some examples are given below.
The detection of PrPSc by immunocytochemical examination of tonsillar biopsies of healthy offspring of scrapie-affected dams has recently been reported (van Keulen et al., 1996). This could, if validated, become a valuable test for use in clinically-suspect live animals, healthy sheep in more vulnerable situations (e.g. imported sheep) and for use in abattoir surveys.
O’Rourke et al. (1998) have reported another potentially useful method by assessing the presence of PrPSc (by immunocytochemistry) in the lymphoid follicles in the nictitating membrane (third eyelid) of sheep with clinical scrapie and some clinically healthy flock-mates of scrapie-affected sheep in 11 flocks with histologically confirmed scrapie. Nine clinical cases and 16 clinically healthy sheep showed positive PrPSc staining. Of these 16 sheep, six subsequently went on to develop scrapie two to seven months later. The remainder were still under observation at the time of the report. The advantage of the method lies in the accessibility of the source tissue that can be biopsied under local anaesthesia in contrast to tonsil biopsy that demands general anaesthesia. Both methods could be used as part of a screening programme using abattoir material. However, more work needs to be done in order to standardise the method and to determine the sensitivity and specificity in a range of breeds and individuals with different PrP genotypes and affected with different agent strains. Studies should of course involve study of tissues from cases of experimental BSE in sheep.
One of the difficulties with immunocytochemical detection of PrP is to distinguish the normal form PrPC from the disease-specific form, PrPSc. The recent development of a monoclonal antibody that specifically detects PrPSc and not PrPC (Korth et al., 1997) will, if validated in practice, significantly aid this process.
Race, Jenny and Sutton (1998) have identified PrPSc in placental cotyledons of pregnant or recently lambed sheep and suggested that this tissue might be useful as a non-invasive method in a scrapie surveillance programme.
Stack et al., 1993 have reported their important findings that SAF can be found in autolysed brain following incubation at 37 degrees C for seven days thus permitting a diagnosis to be made in brain tissue that is quite unsuitable for microscopic examination. The same can be said for such tissue examined by immunoblotting methods (R.E. Race, personal communication). Stack et al., 1995 and 1996 have reported on different electron microscopical methods and biochemical extraction methods to demonstrate SAF and concluded that the introduction of a double homogenization stage and ultrasonic disintegration step are beneficial. Cooley, Clark and Stack (1998) compared immunoblotting for PrP with SAF detection for the diagnosis of natural ovine scrapie and found the former more sensitive. Stack et al. (1998) have reported the detection of SAF in the pituitary gland, in lymph nodes, intestine and spleen of sheep with scrapie thus expanding the range of tissue to which the method can be applied.
Chaplin, Aldrich and Stack, 1998 have developed a method to detect SAF from fixed tissues. There is thus a considerable extension being made to diagnostic methodology and Argentina will be evaluating some of these methods so that effective and efficient diagnoses can be achieved in the varying conditions of practice. Argentina is intending to use some of these methods from now on and to introduce them as part of the formal continuous monitoring and surveillance system once they are validated and internationally agreed.
2.4 The cause
The agents that cause TSE have not been precisely defined. There are three main hypotheses.
The agent is:
Each hypothesis has to explain how mutants can occur and the existence of strains as in conventional agents. The prion hypothesis has much support and has been modified as new discoveries are made. However, it does not so readily explain the existence of strains as do the other two theories which however, lack formal proof of the existence of a conventional genome.
2.5 Strains of agents
There are about 20 biologically defined strains of scrapie in the UK. Definition is achieved by analysis of incubation period and lesion profile data in in-bred strains of mice and an F1 hybrid.
There appears to be only one major strain of the BSE agent isolated from cattle affected with BSE from different geographical locations. The agent isolated from domestic cats with feline spongiform encephalopathy (FSE) (Pearson, et al., 1993), from kudu and nyala with spongiform encephalopathy, from pigs, sheep and goats with experimental BSE appears biologically indistinguishable from the agent that causes BSE, despite considerable differences in the PrP gene sequences in these species (Bruce et al., l994). These findings lend strength to the notion that the scrapie agent has a genome independent of PrP. The BSE agent strain is different from that of any isolated so far from sheep or goats or six cases of historical or contemporary cases of human sporadic CJD in the UK including those isolated from two farmers who each had cases of BSE in their herds. However, the strain of agent isolated from three patients with v-CJD appears to be biologically identical with that isolated from cases of BSE (Bruce et al., l997).
2.6 Molecular strain typing
Studies of the fragment sizes following proteinase K cleavage and glycosylation patterns after Western blotting of PrP in human TSE have been reported by Collinge et al. (1996). Until the advent of v-CJD three types of glycosylation (I-III) had been established by analysis of PrP from different human cases of CJD. All brains from patients with v-CJD fit into a new fourth type (type IV). Furthermore, analysis of the glycosylation of PrP from the brains of a cat with natural FSE, brains of mice and a macaque monkey inoculated with BSE and a cow with natural BSE show a pattern closely similar to type IV. Somerville et al. (1997) have shown that there is a spectrum of patterns obtained that do not fit precisely into rigid classes. Furthermore they showed that the glycosylation pattern was not necessarily perpetuated on passage whereas the biological strain type was, and that historical scrapie isolates were already known to have patterns not too dissimilar to that of type IV yet by conventional (mouse) strain typing the strains are quite different. Other workers (Parchi et al., 1997) also have different views on some of the reported findings but it is clear that at present the method is of value in human CJD to clearly and rapidly distinguish the v-CJD form of PrP from that in other forms of disease.
More recent work reported by Kuczius, Haist and Groschup (1998) in Germany and by Hill et al. (1998) in England suggests that large scale screening of sheep for BSE may be possible. Neither of these studies revealed the characteristic molecular profile of PrP from cattle with BSE in any of the samples from field scrapie of sheep. However, some reservations are held about the method because of inconsistencies in results using the same samples in different laboratories. These may be attributed to differences in methodology that is currently being resolved. Hope et al. (1999) have also reported the results of molecular analyses of PrP from five control sheep and 23 contemporary or archived cases of natural scrapie and experimental scrapie in sheep and experimental BSE in sheep. They found biochemical evidence of strain variation in the PrP analyses from diseased animals. Interestingly they also found at least one isolate of natural scrapie called CH1641 (Foster and Dickinson, 1988) that had a closely similar, but not identical, PrP profile to that of BSE. However, biological strain typing clearly differentiates CH1641 from BSE because this isolate is, in contrast to BSE agent, very difficult to transmit to mice. Thus the authors have reservations about the ability of the methods used to readily distinguish BSE in sheep from the CH1641 strain in sheep, the incidence of occurrence of which in the national flock is unknown. CH1641 was isolated from a Cheviot sheep in 1970 therefore it pre-dates the onset of BSE by about 15 years.
There is no currently harmonised method to characterise animal-derived TSE agent isolates by molecular strain typing. However, clearly the method shows considerable promise and may be developed so it could be used on an international basis to study the epidemiology of the various TSE and eventually may be incorporated as part of a risk assessment procedure . The merit of the method is that it is far quicker than biological strain typing that may take years and, in humans with v-CJD at least, it can be effective on peripheral tissue such as tonsils. (Hill et al., 1997), so it has much to commend it.
The pathogenesis of sheep scrapie is not certainly known but hypothetically may follow that of experimental scrapie in mice (Kimberlin and Walker, 1988, 1989) where, following infection by a peripheral route, including the oral route, infectivity and replication are first detectable in the lymphoreticular system tissues, notably of the spleen, lymph nodes and intestine (Peyer’s patches). The infectivity then enters the thoracic spinal cord via the splanchnic nerve and then travels rostrally to the brain, and caudally to the lumbo-sacral spinal cord.
This sequential pattern of spread was subsequently examined by Beekes, Baldauf and Diringer (1996) and Baldauf, Beekes and Diringer (1997) but using hamsters challenged orally with scrapie infectivity and using a new densitometric method for detecting PrP. The results were conclusive and virtually identical to those reported earlier in mice by Kimberlin and Walker (1988, 1989). However, in addition they provided preliminary evidence that an alternative route of spread to the CNS was via the vagus nerve. In a subsequent publication, Beekes, McBride and Baldauf (1998) conclusively showed that in the hamster model of scrapie induced orally, initial infection of the brain occurs via the vagus nerve.
The following account of the pathogenesis of natural scrapie
in sheep can be surmised from the results of the seminal studies of Hadlow and
co-workers (Hadlow et.al., 1979), who studied the infectivity of a wide
range of tissues collected during the incubation period of Suffolk sheep with
natural scrapie and from Suffolk sheep and goats in the clinical phase of disease
(Hadlow et al., 1980, 1982). Following effective natural exposure of
sheep, presumably by the oral route and, in natural cases due to maternal transmission
by an uncertain route, there is a zero phase, perhaps lasting several months
(e.g. eight months), when infection cannot be detected in any tissue.
Replication is first detected in lymphoreticular system (LRS) tissues, intestine,
lymph nodes and spleen by ten months of age. By about two years of age infectivity
is detected in the CNS. This is followed by replication in the brain in which
high titres of infectivity develop as clinical signs are initiated and progress.
Most other tissues like muscle, heart and milk show no detectable infectivity
whilst others such as bone marrow, pancreas and thymus may show minimal or exceptional
infection. The above outline is not proved in sheep but fits the data from Hadlow
et al. (1980, 1982) and the classical pathogenesis studies of Kimberlin
and Walker (1988, 1989) in mice. From these studies it is possible to divide
tissues into those of high, medium and low risk and distinguish them from those
in which no infection was found. Based on infectivity titres, essentially
Natural scrapie has been diagnosed in sheep, goats and moufflon (Ovis musimon) (Wood and Done, 1992). Experimentally, scrapie has been transmitted to a wide range of mammalian species notably mice and hamsters which make useful models to study the agent and disease. Scrapie has been successfully experimentally transmitted to primates but chimpanzees have so far not succumbed many years after intracerebral inoculation (C.J. Gibbs Jnr., personal communication). Transmission to species other than goats and moufflon has not been recorded as a natural event but sheep scrapie has been incriminated in the origin of BSE via meat-and-bone-meal (Wilesmith et al., 1988 and Wilesmith, Ryan and Atkinson, 1991; Hoinville et.al., 1995). Experimental challenge of cattle and mink with US strains of sheep or goat scrapie via the oral route has not resulted in disease. But intracerebral inoculation of mink will produce transmissible mink encephalopathy (TME) following a longer than natural incubation period. Similarly a neurological disease unlike ‘European BSE’ has been produced by intracerebral challenge of cattle with scrapie agent originating from US sheep and goats (Gibbs et al., 1990; Cutlip et al., 1994, 1997; Robinson, 1996). In this experimental disease microscopic lesions are for the most part mild or absent. PrP is however seen in the brain.
Sheep to sheep transmission by contagion is well known. It is most probably effected via scrapie-infected placenta, presumptively by maternal transmission and also to related and unrelated animals horizontally by the oral route (Pattison et al., 1972, 1974). This is the reason why scrapie is difficult to eradicate and often becomes endemic once introduced, unless draconian methods of control are adopted. It is uncertain whether scrapie is transmitted between goats in these ways. Experimental BSE in goats has been shown not to transmit via the embryo (Foster et al., 1998, 1999). Transmission of experimental scrapie via the sheep embryo is uncertain and different results have been obtained by different workers using different embryo washing methods (Wrathall, 1997).
2.9 Variation in host response to Scrapie
It is generally much harder to transmit scrapie-like diseases between species than within species due to the phenomenon known as the species barrier which has two components, the agent strain and the variation in the PrP gene sequence of the donor and recipient species.
Disease results from the interaction of the agent genome (agent strain) with the host genotype. The PrP gene of sheep is polymorphic in at least four sites, two of which (codons 136 and 171) are the most important in influencing the results of the exposure to the agent. Knowledge of the various combinations of the allelic variation and responses to experimental and natural challenge with scrapie enable judgements to be made of the relative resistance or susceptibility (probably operating via significant control of incubation period length) to particular strains of scrapie. Analysis of the PrP genotype in natural cases of scrapie in sheep shows a clear association with different allelic combinations that may also vary with the breed (Hunter, 1997). This has allowed the use of PrP genotyping to select rams of a "resistant" genotype which increases the distribution of these beneficial alleles in the offspring thus reducing the incidence of scrapie over a few generations (Dawson, 1997). Dawson et al. (1998) have written a concise account of PrP gene variation in sheep and how the results of genotyping can be used in a practical manner to assist in the control of scrapie. Some authors have pointed out dangers in these methods because such sheep may be more susceptible to particular agent strains e.g. BSE. So far however, no problems have been identified in practice. The dose (amount and titre) and route of infection also influence the host response to exposure.
In goats there are also variations in PrP gene structure. One variation consists of the shortest known PrP allele that has only 3 octapeptide repeats but which is non-pathogenic (Goldmann et al., 1998). A dimorphism occurs at codon 142 and this is associated with different incubation periods in goats experimentally inoculated with two strains of scrapie and BSE (Goldmann et al., 1996).
2.10 Is Scrapie an infectious or genetic disease?
There is much evidence to support the view that scrapie is an infectious disease and some to show it may be genetic. When sheep in some flocks in scrapie-affected countries have been examined all sheep with the allelic combination conferring extreme susceptibility have eventually succumbed to scrapie thus suggesting that such sheep were pre-destined to develop scrapie. However, recent studies in sheep from New Zealand, where scrapie is believed to be absent, have shown such susceptible sheep to be devoid of scrapie, even in old age (Hunter et al., 1997; Bossers, Harders & Smits, 1999). This strongly supports the view that scrapie is an infectious disease especially as other reports have indicated that New Zealand sheep can succumb to experimental challenge with scrapie (New Zealand 1997, citing Hourrigan).
2.11 Classification of Scrapie-like diseases
Until 1985 six scrapie-like TSE were known: CJD, Gerstmann Sträussler Scheinker disease and kuru of humans, scrapie of sheep and goats, TME and chronic wasting disease (CWD) of some species of deer and elk.
In humans with CJD three forms were then known: the sporadic form (about 85% of cases) with unknown cause, a rarer familial form (about 10-15% of cases) caused by mutations in the PrP gene and a much rarer iatrogenic form caused by historical medical accidents and due to transmissions from human tissues e.g. pituitary gland via hormones derived therefrom, cornea or dura mater. Sporadic CJD occurs worldwide at a constant incidence of about one case per 1-2 million pa even in countries without BSE. This suggests that the animal and historical human TSE are not epidemiologically connected.
In sheep an iatrogenic form of scrapie occurred in the UK in the late 1930s when a louping-ill vaccine prepared from formalised sheep brains was accidentally contaminated with brains of scrapie affected sheep and several hundred inoculated sheep later succumbed to the disease (Gordon, 1946; Greig, 1950). Other recent incidents of iatrogenic scrapie in small ruminants have been reported from Italy by Cappuchio et al (1998) in goats, and by Agrimi et al. (1999) in sheep and goats. The common vehicle may have been a sub-cutaneously administered vaccine against Myocoplasma agalactiae prepared from brain and mammary tissue of apparently clinically healthy sheep. No other incident of iatrogenic TSE in animals has been found in the literature so it is assumed to be a rare event. There is however no reason why any species of animal could not succumb to a sporadic or familial form of TSE as an exceptionally rare event (< 1 case in 1 million pa). In itself such an incidence is unimportant provided there is no way in which the disease can be recycled within or between species and the agent responsible is not a human pathogen.
PrP is the Prion Protein or (partially) Protease resistant Protein associated with the diseased state. PrPSc is the disease-specific form derived from the normal cellular form PrPC by an unknown mechanism as a post-translational event. The change is in the secondary structure, particularly in 4 domains that are converted from a helix to b sheet (Gabizon and Taraboulos, 1997). A second protein, protein X is now thought to be involved in the conversion process (Safar and Prusiner, 1998). The two forms of protein are easily distinguishable since PrPC is denatured by proteases, such us Proteinase K, whilst PrPSc is partially resistant: after digestion with proteases it yields a resistant fragment of (27-30 kD, PrP27-30). PrPSc and PrP27-30 aggregate to form SAF. Detection of the disease-specific isoform by immunological methods is the basis of several diagnostic tests.