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

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.

Caprine prion gene polymorphisms are associated with decreased incidence of classical scrapie in goat herds in the United Kingdom.

The application of genetic breeding programmes to eradicate transmissible spongiform encephalopathies in goats is an important aim for reasons of animal welfare as well as human food safety and food security. Based on the positive impact of Prnp genetics on sheep scrapie in Europe in the past decade, we have established caprine Prnp gene variation in more than 1100 goats from the United Kingdom and studied the association of Prnp alleles with disease phenotypes in 150 scrapie-positive goats. This investigation confirms the association of the Met142 encoding Prnp allele with increased resistance to preclinical and clinical scrapie. It reveals a novel association of the Ser127 encoding allele with a reduced probability to develop clinical signs of scrapie in goats that are already positive for the accumulation of disease-specific prion protein in brain or periphery. A United Kingdom survey of Prnp genotypes in eight common breeds revealed eleven alleles in over thirty genotypes. The Met142 encoding allele had a high overall mean allele frequency of 22.6%, whereas the Ser127 encoding allele frequency was considerably lower with 6.4%. In contrast, a well known resistance associated allele encoding Lys222 was found to be rare (0.9%) in this survey. The analysis of Prnp genotypes in Mexican Criollas goats revealed nine alleles, including a novel Phe to Leu substitution in codon 201, confirming that high genetic variability of Prnp can be found in scrapie-free populations. Our study implies that it should be feasible to lower scrapie prevalence in goat herds in the United Kingdom by genetic selection.

Quantification of Peyer's patches in Cheviot sheep for future scrapie pathogenesis studies.

Peyer's patches (PPs) are the most probable sites of intestinal uptake of the transmissible spongiform encephalopathy (TSE) agent. The amount of PP tissue varies considerably between different age groups of individuals, and whether this variation is related to susceptibility to TSE infection raises an intriguing possibility. The purpose of this study was to determine the surface area of PP tissue and the number of associated lymphoid follicles in different age groups of Neuropathogenesis Unit (NPU) Cheviot sheep. Terminal ilea were obtained from 33 sheep of different ages. Samples of ileal tissue were collected for immunocytochemistry and immunolabelled for prion protein (PrP). Specimens were then fixed in acetic acid, stained with methylene blue and transilluminated. Image analysis software was used to calculate the area of intestinal and PP tissue. The number of associated lymphoid follicles was determined using a dissecting microscope. Results showed a marked fall in surface area of PP tissue and lymphoid follicle density around puberty (about 8-9 months of age in NPU Cheviot sheep) and both measures remained low throughout adulthood. Using the Spearman's rank correlation coefficient, r(s), these two measures were found to be closely correlated (r(s)=0.899, n=33, P<0.0001). There was also a significant (negative) correlation between age and the two respective measures (surface area of PP tissue versus age, r(s)=-0.879 (n=33, P<0.0001); lymphoid follicle density versus age r(s)=-0.943 (n=33, P<0.0001). Immunolabelling for PrP was observed primarily in the light zone of lymphoid follicles. Results obtained from this study are useful for future oral pathogenesis studies of the NPU Cheviot flock. They may also offer a possible biological explanation for the apparent age-susceptibility relationship observed in natural cases of TSEs and might help to explain the young age-distribution of cases.

Partial dissociation of PrP(Sc) deposition and vacuolation in the brains of scrapie and BSE experimentally affected goats.

The diagnosis of transmissible spongiform encephalopathies (TSEs) depends on the detection of vacuolation in brain sections taken from affected individuals and/or the identification of the disease-associated isoform of the PrP (prion) protein (PrP(Sc)). During the course of an investigation, goats clinically affected following experimental infection with three different sources of TSE (SSBP/1, CH1641 and BSE) developed widespread vacuolar degeneration in the brain. With BSE, PrP(Sc) was clearly recognized in affected goat brain by immunocytochemistry (icc) and Western blotting, but in contrast the experimental scrapie sources SSBP/1 and CH1641 showed almost no or very little PrP(Sc) by icc. Western blot analysis of PrP(Sc) from BSE-affected and SSBP/1-affected goat brain showed that the protein was present in brain affected by both TSE sources, but could not be used to determine how much protein was present. It became clear that PrP(Sc) and vacuolation could be partially dissociated following challenge with two of the three TSE sources. Subtle differences in glycosylation patterns between BSE- and SSBP/1-associated PrP protein isoforms could also be recognized, although these experimentally generated results should not be regarded as a BSE/scrapie differential test. However, our study warns that the reliance on PrP(Sc) determination by icc alone as a means by which to diagnose TSE infection may generate false negative results.

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.