Fiona Houston

Transmission of prion diseases by blood transfusion

Attempts to detect infectivity in the blood of humans and animals affected with transmissible spongiform encephalopathies (TSEs or prion diseases) have often been inconclusive because of the limitations of cross-species bioassays and the small volumes of blood that can be injected by the intracerebral route. A model has been developed for the experimental study of TSE transmission by blood transfusion using sheep experimentally infected with bovine spongiform encephalopathy (BSE) or natural scrapie as donors and susceptible scrapie-free sheep as recipients. Donors and recipients of the same species greatly increase the sensitivity of the bioassay and in sheep large volumes of blood can be injected by the intravenous (i.v.) route. Transmission of BSE to a single animal using this approach was reported recently. This study confirms this result with a second transmission of BSE and four new cases of transmission of natural scrapie. Positive transmissions occurred with blood taken at pre-clinical and clinical stages of infection. Initial studies indicate that following such infection by the i.v. route, deposition of the abnormal prion protein isoform, PrP(Sc), in peripheral tissues may be much more limited than is seen following oral infection. These results confirm the risks of TSE infection via blood products and suggest that the measures taken to restrict the use of blood in the UK have been fully justified.

Prion diseases are efficiently transmitted by blood transfusion in sheep

The emergence of variant Creutzfeld-Jakob disease, following on from the bovine spongiform encephalopathy (BSE) epidemic, led to concerns about the potential risk of iatrogenic transmission of disease by blood transfusion and the introduction of costly control measures to protect blood supplies. We previously reported preliminary data demonstrating the transmission of BSE and natural scrapie by blood transfusion in sheep. The final results of this experiment, reported here, give unexpectedly high transmission rates by transfusion of 36% for BSE and 43% for scrapie. A proportion of BSE-infected transfusion recipients (3 of 8) survived for up to 7 years without showing clinical signs of disease. The majority of transmissions resulted from blood collected from donors at more than 50% of the estimated incubation period. The high transmission rates and relatively short and consistent incubation periods in clinically positive recipients suggest that infectivity titers in blood were substantial and/or that blood transfusion is an efficient method of transmission. This experiment has established the value of using sheep as a model for studying transmission of variant Creutzfeld-Jakob disease by blood products in humans.

Scrapie epidemic in a fully PrP-genotyped sheep flock

In scrapie-affected sheep flocks, host PrP genotype plays a vital role in determining which sheep will succumb to scrapie and the incubation period. Consequently, within-flock scrapie dynamics is best understood within the context of the genotype profile of the flock. Here we describe a 17 month epidemic of scrapie in a commercially farmed flock of 230 genotyped Texel sheep. At the start of the study, 70% of the sheep were of three genotypes only: ARR/ARQ, ARH/ARQ and ARQ/ARQ. Only 15% of sheep encoded the disease-associated VRQ allele and only a single sheep (0.4%) was of the most susceptible VRQ/VRQ genotype. For susceptible genotypes there was a marked deficit (P<0.025) of older animals (> or =3 years), implying that some cases of scrapie had occurred previously. In the ensuing 17 months, 18 sheep of known genotype were confirmed positive for the disease: seven VRQ/ARQ, six VRQ/ARH, two VRQ/ARR, three ARQ/ARQ. Median ages at death were 2.7, 2.8, 4.2 and 3.8 years respectively. Mortality rates were 55, 86, 13 and 3% respectively. Survival analysis revealed a highly significant effect of genotype on survivorship, but no difference between VRQ/ARQ and VRQ/ARH, or between VRQ/ARR and ARQ/ARQ. There was no difference in the survivorship of middle- and older-age cohorts of susceptible sheep. Scrapie risk group (as defined by PrP genotype) was not associated with submission as a scrapie suspect but later found to be negative, or with dying of unknown causes on the farm.

The signature of scrapie: differences in the PrP genotype profile of scrapie–affected and scrapie-free UK sheep flocks

The amino–acid sequence of the PrP protein plays an important role in determining whether sheep are susceptible to scrapie. Although the genetics of scrapie susceptibility are now well understood, there have been few studies of the PrP gene at the population level, especially in commercially farmed sheep. Here we describe the PrP genetic profiles of the breeding stock of four UK sheep flocks, comprising nearly 650 animals in total. Two flocks had been scrapie affected for about eight years and two were scrapie free. Scrapie-resistant PrP genotypes predominated in all flocks but highly susceptible genotypes were present in each case. The distribution of PrP genotypes was similar in the scrapie-affected and scrapie-free flocks. The former, however, showed a slight but significant skew towards more susceptible genotypes despite their previous losses of susceptible sheep. Surprisingly, this skew was apparent in younger, but not older, sheep. We suggest that these patterns may occur if sheep flocks destined to become scrapie affected are predisposed by a genetic profile skewed towards susceptibility. The age structure of the scrapie-affected flocks suggests that the number of losses attributable directly or indirectly to scrapie considerably exceeds that recognized by the farmers, and also that significant losses may occur even in sheep of a moderately susceptible genotype. Similar patterns were not detected in the scrapie-free flocks, indicating that these losses are associated with scrapie infection as well as genotype.

Susceptibility of young sheep to oral infection with bovine spongiform encephalopathy decreases significantly after weaning.

ABSTRACT Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy (TSE) (or prion disease) that is readily transmissible to sheep by experimental infection and has the shortest incubation period in animals with the ARQ/ARQ PRNP genotype (at codons 136, 154, and 171). Because it is possible that sheep in the United Kingdom could have been infected with BSE by being fed contaminated meat and bone meal supplements at the same time as cattle, there is considerable interest in the responses of sheep to BSE inoculation. Epidemiological evidence suggests that very young individuals are more susceptible to TSE infection; however, this has never been properly tested in sheep. In the present study, low doses of BSE were fed to lambs of a range of ages (∼24 h, 2 to 3 weeks, 3 months, and 6 months) and adult sheep. The incidence of clinical BSE disease after inoculation was high in unweaned lambs (∼24 h and 2 to 3 weeks old) but much lower in older weaned animals The incubation period was also found to be influenced by the genotype at codon 141 of the PRNP gene, as lambs that were LF heterozygotes had a longer mean incubation period than those that were homozygotes of either type. The results suggest that sheep in the United Kingdom would have been at high risk of BSE infection only if neonatal animals had inadvertently ingested contaminated supplementary foodstuffs.

Pathogenetical significance of porencephalic lesions associated with intracerebral inoculation of sheep with the bovine spongiform encephalopathy (BSE) agent

Decreased rates of transmission of transmissible spongiform encephalopathies (TSEs) to sheep have been attributed to some polymorphisms of the prion protein (PrP) and to a ‘species barrier’ on interspecies experiments. In addition, the blood–brain barrier may be a further impediment to TSE neuroinvasion. The intracerebral (I/C) route is generally considered the most efficient for TSE transmission, as it may help to bypass those factors. Therefore, susceptibility of particular species to specific TSE agents is conducted by this route. Aims: This study characterizes the traumatic brain lesions associated with the I/C injection of the bovine spongiform encephalopathy agent in sheep, assesses the relevance of such lesions in the outcome of clinical disease and provides insight into the mechanisms of PrPd conversion and amplification following I/C challenge. Methods: A total of 27 hemibrains have been macroscopically and immunohistochemically examined to investigate the presence of lesions compatible with the needle track and the PrPd distribution, respectively. Results: No residual inoculum was found and the extension and severity of the traumatic brain lesions were unrelated to the clinical outcome. Sheep with PrPd accumulation in the brain also showed conspicuous focal aggregates in the porencephalic lesions and in the circumventricular organs. In contrast, sheep without PrPd deposits in the brain were also negative in the traumatic lesions. Conclusion: Overall, these findings suggest that the efficiency of the I/C route is due to effective absorption and blood recirculation of infection, rather than to primary amplification at the site of injection.

Comparative Susceptibility of Sheep of Different Origins, Breeds and PRNP Genotypes to Challenge with Bovine Spongiform Encephalopathy and Scrapie

Sheep are natural hosts of the prion disease, scrapie. They are also susceptible to experimental challenge with various scrapie strains and with bovine spongiform encephalopathy (BSE), which affects cattle and has been accidentally transmitted to a range of other species, including man. Incidence and incubation period of clinical disease in sheep following inoculation is controlled by the PRNP gene, which has different alleles defined on the basis of polymorphisms, particularly at codons 136, 154 and 171, although other codons are associated with survival time, and the exact responses of the sheep may be influenced by other breed-related differences. Here we report the results of a long term single study of experimental scrapie and BSE susceptibility of sheep of Cheviot, Poll Dorset and Suffolk breeds, originating from New Zealand and of a wide range of susceptible and resistant PRNP genotypes. Responses were compared with those of sheep from a closed Cheviot flock of UK origin (Roslin Cheviot flock). The unusually long observation period (6–8 years for most, but up to 12 years for others) allows us to draw robust conclusions about rates of survival of animals previously regarded as resistant to infection, particularly PRNP heterozygotes, and is the most comprehensive such study reported to date. BSE inoculation by an intracerebral route produced disease in all genotype groups with differing incubation periods, although M112T and L141F polymorphisms seemed to give some protection. Scrapie isolate SSBP/1, which has the shortest incubation period in sheep with at least one VRQ PRNP allele, also produced disease following sub-cutaneous inoculation in ARQ/ARQ animals of New Zealand origin, but ARQ/ARQ sheep from the Roslin flock survived the challenge. Our results demonstrate that the links between PRNP genotype and clinical prion disease in sheep are much less secure than previously thought, and may break down when, for example, a different breed of sheep is moved into a new flock.

Pathological Phenotype of Sheep Scrapie After Blood Transfusion

Blood transfusion practices have resulted in iatrogenic cases of variant Creutzfeldt-Jakob disease (vCJD) and it is known that sheep blood is also infectious in the pre-clinical stages of natural scrapie and experimentally induced bovine spongiform encephalopathy (BSE). Further investigations have also shown that the pathological phenotype of sheep BSE and human vCJD is maintained after blood transfusion. The present study describes the pathological phenotype, in terms of accumulation of the disease-associated prion protein in brain and lymphoreticular tissues, in sheep receiving blood from donors infected with natural scrapie. The immunohistochemical examinations undertaken showed a degree of phenotypic variability within and between scrapie donors and recipients, which might be attributable to the presence of more than one scrapie strain amongst the donor sheep or to a host adaptation process, or to the interaction of both, rather than to the influence of the route of infection.

Use of a preclinical test in the control of classical scrapie

Scrapie control in Great Britain (GB) was originally based on the National Scrapie Plans Ram Genotyping scheme aimed at reducing the susceptibility of the national flock. The current official strategy to control scrapie in the national flock involves culling susceptible genotypes in individual, known affected flocks (compulsory scrapie flock scheme or CSFS). However, the recent development of preclinical test candidates means that a strategy based on disease detection may now be feasible. Here, a deterministic within-flock model was used to demonstrate that only large flocks with many home-bred ewes are likely to be a significant risk for flock-to-flock transmission of scrapie. For most other flocks, it was found that the CSFS could be replaced by a strategy using a currently available live test without excessive risk to other farmers, even if the proportion of susceptible genotypes in the flock is unusually large. Even for flocks that represent a high risk of harbouring a high prevalence of infection, there would be limited probability of onward transmission if scrapie is detected soon after disease introduction (typically less than 5 years). However, if detection of disease is delayed, the existing CSFS strategy may be the most appropriate control measure in these cases.

An Economic Evaluation of Preclinical Testing Strategies Compared to the Compulsory Scrapie Flock Scheme in the Control of Classical Scrapie

Cost-benefit is rarely combined with nonlinear dynamic models when evaluating control options for infectious diseases. The current strategy for scrapie in Great Britain requires that all genetically susceptible livestock in affected flocks be culled (Compulsory Scrapie Flock Scheme or CSFS). However, this results in the removal of many healthy sheep, and a recently developed pre-clinical test for scrapie now offers a strategy based on disease detection. We explore the flock level cost-effectiveness of scrapie control using a deterministic transmission model and industry estimates of costs associated with genotype testing, pre-clinical tests and the value of a sheep culled. Benefit was measured in terms of the reduction in the number of infected sheep sold on, compared to a baseline strategy of doing nothing, using Incremental Cost Effectiveness analysis to compare across strategies. As market data was not available for pre-clinical testing, a threshold analysis was used to set a unit-cost giving equal costs for CSFS and multiple pre-clinical testing (MT, one test each year for three consecutive years). Assuming a 40% within-flock proportion of susceptible genotypes and a test sensitivity of 90%, a single test (ST) was cheaper but less effective than either the CSFS or MT strategies (30 infected-sales-averted over the lifetime of the average epidemic). The MT strategy was slightly less effective than the CSFS and would be a dominated strategy unless preclinical testing was cheaper than the threshold price of £6.28, but may be appropriate for flocks with particularly valuable livestock. Though the ST is not currently recommended, the proportion of susceptible genotypes in the national flock is likely to continue to decrease; this may eventually make it a cost-effective alternative to the MT or CSFS.