J. Foster

Transmission of BSE by blood transfusion in sheep

We have shown that it is possible to transmit bovine spongiform encephalopathy (BSE) to a sheep by transfusion with whole blood taken from another sheep during the symptom-free phase of an experimental BSE infection. BSE and variant Creutzfeldt-Jakob disease (vCJD) In human beings are caused by the same infectious agent, and the sheep-BSE experimental model has a similar pathogenesis to that of human vCJD. Although UK blood transfusions are leucodepleted--a possible protective measure against any risk from blood transmission--this report suggests that blood donated by symptom-free vCJD-infected human beings may represent a risk of spread of vCJD infection among the human population of the UK.

Natural scrapie in a closed flock of Cheviot sheep occurs only in specific PrP genotypes

SummaryNatural scrapie in a closed flock of South Country Cheviot sheep has resulted in 45 deaths between 1986 and 1995. Of these cases, 35 sheep have been analysed for disease-linked PrP gene polymorphisms and all encode valine at codon 136 on at least one allele with 77% homozygous (VV136) and 23% valine/alanine heterozygotes (VA136). Mean survival time was 907 and 1482 days for VV136 and VA136 scrapie affected animals respectively. VV136 animals were all at great risk of disease if allowed to live long enough. However scrapie occurred only in a specific subgroup of VA136 sheep, survival advantage depending on VA136 animals being heterozygous for other polymorphisms at codons 154 or 171. The flock history has been recorded in great detail since its foundation in 1960 however there was no strong evidence for simple maternal or paternal transmission of disease other than inheritance of PrP genotype.

Natural scrapie and PrP genotype: case-control studies in British sheep.

Natural scrapie in sheep is associated with polymorphisms of the PrP gene, particularly at amino acid codons 136, 154 and 171. This paper reports the results of nine scrapie case-control studies in Bleu du Maine, Herdwick, Merino x Shetland, Poll Dorset, Scottish Halfbred, Shetland, Soay, Suffolk and Swaledale sheep from British flocks affected by scrapie. In some outbreaks, scrapie was found to occur only in animals with at least one PrP allele encoding valine at codon 136 (V136), usually a relatively rare allele in healthy controls In other outbreaks, the v136, PrP aUlele was either not found or was not an absolute prerequisite for scrapie to develop. Although scrapie had a strong tendency to affect sheep with PrP genotypes homozygous for glutamine at codon 171 (QQ136), these genotypes (QQ136 but varying at other codon positions) were relatively common in healthy controls. The reliable prediction of scrapie susceptibility in previously uninvestigated sheep flocks will therefore require information at least about PrP genotypes at codons 136 and 171.

Clinical signs, histopathology and genetics of experimental transmission of BSE and natural scrapie to sheep and goats.

This paper compares the clinical signs, histopathology, detection of PrPSc protein and PrP genetics of the transmission of BSE to sheep and goats, with the effects of the transmission of natural scrapie from a brain homogenate from a single sheep. After intracerebral and oral inoculations there were similarities in the clinical signs due to the two sources of infection, but there were differences in pathology at the end stage of disease and in the genotypes of the sheep which succumbed to the challenges. The incubation period of BSE was associated with the sheep PrP codon 171 genotype, but the natural scrapie source, despite inducing disease only in known susceptible genotypes, showed no clear association with PrP genotype.

Distribution of the prion protein in sheep terminally affected with BSE following experimental oral transmission.

This study has examined the distribution of PrPSc in sheep by immunocytochemistry of tissues recovered from terminally affected animals following their experimental infection by the oral route with BSE. Despite a wide range of incubation period lengths, affected sheep showed a similar distribution of high levels of PrPSc throughout the central nervous system. PrPSc was also found in the lymphoid system, including parts of the digestive tract, and some components of the peripheral nervous system. These abundant PrPSc deposits in sheep in regions outside the central nervous system are in direct contrast with cattle infected with BSE, which show barely detectable levels of PrPSc in peripheral tissues. A number of genetically susceptible, challenged animals appear to have survived.

Modelling the spread of scrapie in a sheep flock: evidence for increased transmission during lambing seasons

Summary.Presence of scrapie infectivity in the placenta suggests the possibility of increased transmission of scrapie during the lambing season. This hypothesis was explored here using a mathematical model of scrapie transmission dynamics which has previously been successfully used to study several scrapie outbreaks in Scottish sheep flocks. It was applied here to the Langlade experimental sheep flock (INRA Toulouse, France), in which a natural scrapie epidemic started in 1993. Extensive data were available, including pedigree, scrapie histopathological diagnoses and PrP genotypes. Detailed simulations of the scrapie outbreak reveal that the observed patterns of seasonality in incidence can not be accounted for by seasonality in demography alone and provide strong support for the hypothesis of increased transmission during lambing. Observations from several other scrapie outbreaks also showing seasonal incidence patterns support these conclusions.

Nor98-like sheep scrapie in the United Kingdom in 1989

THE atypical form of scrapie in sheep, termed Nor98, was first described in clinically affected Norwegian sheep diagnosed from 1998 onwards (Benestad and others 2003). Nor98 is characterised by its atypical neuropathology and by the unusual banding pattern seen in Western blots of the pathological prion protein, PrPSc. Since Nor98 was first recognised, cases with similar features have been identified in several other countries, including the UK, mostly in the course of active surveillance of asymptomatic sheep (Buschmann and others 2004, De Bosschere and others 2004, Gavier-Widen and others 2004, Onnasch and others 2004, Orge and others 2004, Epstein and others 2005, Everest and others 2006). This short communication describes a clinical case of sheep scrapie with the distinguishing features of Nor98 that occurred in the UK in 1989. A Dorset horn sheep was presented to the Moredun Research Institute, Edinburgh, to confirm a clinical diagnosis of scrapie; records of the source flock and details of the clinical presentation are no longer available. Formalin-fixed and frozen brain samples were passed on to the Neuropathogenesis Unit for further investigation. The occurrence and incubation period of scrapie in sheep is controlled by the host gene encoding PrP (Hunter 1997). Many polymorphisms of this gene have been identified, but the major amino acid substitutions influencing scrapie are at codons 136 (A/V), 154 (R/H) and 171 (Q/R/H). The PrP genotype of the Dorset horn sheep at these three codons was A136H154Q171/A136H154Q171 (AHQ/AHQ), a genotype that confers relative but not absolute resistance to ‘classical’ scrapie in the UK (Baylis and others 2004). However, cases of Nor98 scrapie in Norway have been significantly associated with the presence of the AHQ allele (Moum and others 2005). Neuropathological changes indicative of scrapie were seen predominantly in the cerebellum. This pathology consisted of a pronounced vacuolar degeneration of both the molecular and granular layers, accompanied by an abnormal accumulation of PrP (Fig 1); the changes were most severe in the granular layer. Milder pathological changes were seen in some brainstem nuclei, but areas adjacent to the obex, such as the dorsal motor nucleus of the vagus, were unaffected. The cerebral cortex showed only very mild pathology. This neuropathological distribution differs from that seen in most cases of classical scrapie, but, apart from the relative sparing of the cerebrum, resembles that seen in Nor98 scrapie (Benestad and others 2003). Protease-resistant PrPSc from the Dorset horn sheep was analysed by Western blotting and compared with PrPSc from cases of classical scrapie (in an ARQ/AHQ sheep) and Nor98 (in ARQ/AHQ and AHQ/AHQ sheep) (Fig 2). Brain tissues were homogenised in 100mM Tris-HCl (pH 7·4) with 2 per cent N-lauroylsarcosine sodium (Sarcosyl; Sigma), digested with proteinase K (200 μg/ml for one hour) and centrifuged at 20,000 g. The pellets were denatured in Laemmli buffer and loaded (3 mg tissue equivalents per lane) on 12 per cent Bis-Tris gel (Invitrogen). The blot was revealed by immunostaining with P4 monoclonal antibody. The Dorset horn isolate showed a PrPSc pattern very similar to the two Nor98 samples but very different from the classical scrapie sample. The Nor98-like pattern with P4 was characterised by the presence of multiple PrPSc fragments in the 12 to 30 kDa molecular weight range, with the 12 kDa fragments giving the strongest signal. In contrast, the classical scrapie isolate showed the typical three PrPSc bands, ranging from 18 to 30 kDa. The atypical phenotype seen in sheep with Nor98 scrapie suggests that they are infected with an unusual strain of scrapie. Scrapie strains can be characterised and compared by transmission studies in rodents, in which each strain produces a unique disease profile. The Nor98-like case described here was included in a study reported by Bruce and others (2002), in which transmissions to mice were attempted from 10 scrapieaffected sheep. The transmission from the Dorset horn sheep, referred to as SCR2 in the paper by Bruce and others (2002), was almost completely negative; this precluded any positive strain characterisation, as scrapie in general transmits poorly to mice. Brain tissue from the Dorset horn sheep was also inoculated into bank voles (Clethrionomys glareolus), together Veterinary Record (2007) 160, 665-666

Population dynamics of a scrapie outbreak.

Summary.u2002A detailed analysis of a scrapie outbreak in a flock of Cheviot sheep is described. A total of 33 cases of 1473 sheep born to the flock were reported between 1985 and 1994. The epidemiology of scrapie can only be understood with reference to sheep demography, the population genetics of susceptibility to scrapie, pathogenesis during a long incubation period, and the rate of transmission (by both horizontal and vertical routes), all of which interact in complex ways. In recent work a mathematical model incorporating these elements was developed and successfully reproduced key features of an earlier outbreak of scrapie in this flock. Here an application of the model to the second outbreak is described. The model accurately reproduces observed allele frequencies and total numbers of susceptible animals remaining at the end of the outbreak. A major difference between the two outbreaks is the very much lower force of infection in the second outbreak. This provided additional information which suggested two ways in which our existing assumptions be refined; firstly, older animals have reduced susceptibility to scrapie and secondly, homozygous and heterozygous susceptibles have different incubation periods.

Comparative epidemiology of scrapie outbreaks in individual sheep flocks.

Data recording the course of scrapie outbreaks in 4 sheep flocks (2 in Cheviot sheep and 2 in Suffolks) are compared. For each outbreak the data on scrapie incidence and sheep demography and pedigrees cover periods of years or decades. A key finding is that the incidence of clinical cases peaks in sheep 2-3 years old, despite very different forces-of-infection. This is consistent with age-specific susceptibility of sheep to scrapie, as has been reported for cattle to bovine spongiform encephalopathy and for humans to variant Creutzfeldt-Jakob disease. Scrapie incidence was higher in ewes than rams and at certain times of years, though these effects were not consistent between flocks. There was no evidence for high levels of vertical transmission.

Derivation of a scrapie-free sheep flock from the progeny of a flock affected by scrapie

The Cheviot flock at the Institute for Animal Healths Neuropathogenesis Unit (npu) has endemic scrapie, which affects primarily vrq/vrq sheep and at high frequency. A new flock with a full range of PrP genotypes, including the highly susceptible vrq/vrq, has been produced on a separate site, from animals in the npu breeding flock, and it remains scrapie-free after eight years. In contrast, in a parallel flock at the npu farm, scrapie has reappeared after five years, although the animals were kept in separate accommodation from the scrapie-affected sheep. During this time the npu breeding flock continued to have scrapie cases. Although it is known that highly susceptible sheep can remain free of infection in a clean environment, this is the first report of the infection being removed successfully from the bloodlines of scrapie-affected sheep. The results confirm that scrapie is not a genetic disease dependent only on the PrP gene sequence, but requires both genetic susceptibility and an infectious agent.