[Show abstract][Hide abstract] ABSTRACT: The role of blood in the iatrogenic transmission of transmissible spongiform encephalopathy (TSE) or prion disease has become an increasing concern since the reports of variant Creutzfeldt-Jakob disease (vCJD) transmission through blood transfusion from humans with subclinical infection. The development of highly sensitive rapid assays to screen for prion infection in blood is of high priority in order to facilitate the prevention of transmission via blood and blood products. In the present study we show that PrP(sc), a surrogate marker for TSE infection, can be detected in cells isolated from the blood from naturally and experimentally infected sheep by using a rapid ligand-based immunoassay. In sheep with clinical disease, PrP(sc) was detected in the blood of 55% of scrapie agent-infected animals (n = 80) and 71% of animals with bovine spongiform encephalopathy (n = 7). PrP(sc) was also detected several months before the onset of clinical signs in a subset of scrapie agent-infected sheep, followed from 3 months of age to clinical disease. This study confirms that PrP(sc) is associated with the cellular component of blood and can be detected in preclinical sheep by an immunoassay in the absence of in vitro or in vivo amplification.
Journal of Virology 10/2009; 83(23):12552-8. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sheep with an ARQ/ARQ PRNP genotype at codon positions 136/154/171 are highly susceptible to experimental infection with bovine spongiform encephalopathy (BSE). However, a number of sheep challenged orally or intracerebrally with BSE were clinically asymptomatic and found to survive or were diagnosed as BSE-negative when culled. Sequencing of the full PRNP gene open reading frame of BSE-susceptible and -resistant sheep indicated that, in the majority of Suffolk sheep, resistance was associated with an M112T PRNP variant (TARQ allele). A high proportion (47 of 49; 96%) of BSE-challenged wild-type (MARQ/MARQ) Suffolk sheep were BSE-infected, whereas none of the 20 sheep with at least one TARQ allele succumbed to BSE. Thirteen TARQ-carrying sheep challenged with BSE are still alive and some have survival periods equivalent to, or greater than, reported incubation periods of BSE in ARR/ARR and VRQ/VRQ sheep.
Journal of General Virology 08/2009; 90(Pt 10):2569-74. · 3.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The potential spread of prion infectivity in secreta is a crucial concern for prion disease transmission. Here, serial protein misfolding cyclic amplification (sPMCA) allowed the detection of prions in milk from clinically affected animals as well as scrapie-exposed sheep at least 20 months before clinical onset of disease, irrespective of the immunohistochemical detection of protease-resistant PrP(Sc) within lymphoreticular and central nervous system tissues. These data indicate the secretion of prions within milk during the early stages of disease progression and a role for milk in prion transmission. Furthermore, the application of sPMCA to milk samples offers a noninvasive methodology to detect scrapie during preclinical/subclinical disease.
Journal of Virology 07/2009; 83(16):8293-6. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During the 1980s, bovine spongiform encephalopathy (BSE)-contaminated meat and bonemeal were probably fed to sheep, raising concerns that BSE may have been transmitted to sheep in the UK. The human disease, variant Creutzfeldt-Jakob disease, arose during the BSE epidemic, and oral exposure of humans to BSE-infected tissues has been implicated in its aetiology. The concern is that sheep BSE could provide another source of BSE exposure to humans via sheep products. Two immunological techniques, Western immunoblotting (WB) and immunohistochemistry (IHC), have been developed to distinguish scrapie from cases of experimental sheep BSE by the characteristics of their respective abnormal, disease-associated prion proteins (PrP(d)). This study compares the WB and IHC characteristics of PrP(d) from brains of primary, secondary and tertiary experimental ovine BSE cases with those of cattle BSE and natural sheep scrapie. Discrimination between experimental sheep BSE and scrapie remained possible by both methods, regardless of the route of challenge.
Journal of General Virology 04/2009; 90(Pt 3):764-8. · 3.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A key event in transmissible spongiform encephalopathies (TSEs) is the conversion of the soluble, protease-sensitive glycosylated prion protein (PrP(C)) to an abnormally structured, aggregated and partially protease-resistant isoform (PrP(Sc)). Both PrP isoforms bear two potential glycosylation sites and thus in a typical western blot with an anti-PrP antibody three distinct bands appear, corresponding to the di-, mono- or unglycosylated forms of the protein. The relative intensity and electrophoretic mobility of the three bands are characteristic of each TSE strain and have been used to discriminate between them.
In the present study we used lectin-based western blotting to evaluate possible variations in composition within sugar chains carried by PrP(Sc) purified from subjects affected with different TSEs. Our findings indicate that in addition to the already well-documented differences in electrophoretic mobility and amounts of the glycosylated PrP(Sc) forms, TSE strains also vary in the abundance of specific N-linked sugars of the PrP(Sc) protein.
These results imply that PrP glycosylation might fine-tune the conversion of PrP(C) to PrP(Sc) and could play an accessory role in the appearance of some of the characteristic features of TSE strains. The differences in sugar composition could also be used as an additional tool for discrimination between the various TSEs.
PLoS ONE 02/2009; 4(5):e5633. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Samples of tissue from the central nervous system (cns), the lymphoreticular system (lrs) and the rectal mucosa of a large number of scrapie-exposed sheep, with and without signs of clinical disease, were examined immunohistochemically for evidence of disease-associated prion protein (PrP(d)). The rectal mucosa has received almost no attention so far in scrapie diagnosis, despite its abundant rectoanal mucosa-associated lymphoid tissue, and its accessibility. The scrapie-confirmed cases included 244 with clinical disease, of which 237 (97.1 per cent) were positive in the rectal mucosa, and 121 apparently healthy sheep, of which 104 (86 per cent) were positive in the rectal mucosa. PrP(d) was detected in 86.4 to 91.5 per cent of the other lrs tissues of the healthy sheep examined and in 77.7 per cent of their cns tissues. The stage of infection, therefore, affected the probability of a positive result in the rectal mucosa, whereas the breed, PrP genotype, age and sex had little or no independent effect. Accumulations of PrP(d) were observed in the rectal mucosa and other lrs tissues of vrq/arr sheep with preclinical and clinical scrapie, albeit with a lower frequency and magnitude than in sheep of other PrP genotypes. Western immunoblotting analyses of samples of rectal mucosa gave the characteristic PrP glycoprofile, with a sensitivity similar to that of immunohistochemistry.
The Veterinary record 04/2006; 158(10):325-31. · 1.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In view of the established link between bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease and of the susceptibility of sheep to experimental BSE, the detection of potential cases of naturally occurring BSE in sheep has become of great importance. In this study, the immunohistochemical (IHC) phenotype of disease-associated prion protein (PrP(d)) accumulation has been determined in the brain of 64 sheep, of various breeds and PrP genotypes, that had developed neurological disease after experimental BSE challenge with different inocula by a range of routes. Sheep BSE was characterized by neuron-associated intra- and extracellular PrP(d) aggregates and by conspicuous and consistent deposits in the cytoplasm of microglia-like cells. The stellate PrP(d) type was also prominent in most brain areas and marked linear deposits in the striatum and midbrain were distinctive. Sheep of the ARR/ARR and ARQ/AHQ genotypes displayed lower levels of PrP(d) than other sheep, and intracerebral BSE challenge resulted in higher levels of PrP(d) accumulating in the brain compared with other routes. The PrP genotype and the route of challenge also appeared to affect the incubation period of the disease, giving rise to complex combinations of magnitude of PrP(d) accumulation and incubation period. Despite these differences, the phenotype of PrP(d) accumulation was found to be very consistent across the different factors tested (notably after subpassage of BSE in sheep), thus highlighting the importance of detailed IHC examination of the brain of clinically affected sheep for the identification of potential naturally occurring ovine BSE.
Journal of General Virology 04/2005; 86(Pt 3):827-38. · 3.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sixty Romney sheep of three prion protein genotypes were dosed orally at six months of age with an inoculum prepared from the brains of cattle clinically affected with BSE, and 15 sheep were left undosed as controls. They were randomly assigned within genotype to groups and were sequentially euthanased and examined postmortem at intervals of six or 12 months, depending on their predicted susceptibility. Tissue pools prepared from the three, four or five dosed animals in each group were inoculated into groups of 20 RIII mice as a bioassay for infectivity. Separate inocula were prepared from the matched control sheep killed at each time. In the ARQ/ARQ sheep killed four months after inoculation, infectivity was detected in the Peyer's patch tissue pool, and at 10 months it was detected in the spleen pool; from 16 months, infectivity was detected in a range of nervous and lymphoreticular tissues, including the spinal cord pool, distal ileum excluding Peyer's patches, liver, Peyer's patches, mesenteric and prescapular lymph nodes, spleen, tonsil and cervical thymus. No infectivity was detected in the tissue pools from the ARQ/ARR and ARR/ARR sheep killed 10 months or 22 months after infection.
The Veterinary record 03/2005; 156(7):197-202. · 1.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Scrapie free adult sheep were introduced to a sheep flock specifically maintained to maximise scrapie infection. Native born sheep of the highly susceptible VRQ/VRQ genotype in this flock show highly efficient transmission, evidenced by 100% infection, with an age at death of less than 2 years. Infection in introduced sheep was identified by biopsy of tonsilar and nictitating membrane lymphoid tissue. Progeny of these sheep were monitored and clinical disease confirmed by examination of the brain using routine diagnostic methods. Naïve sheep of New Zealand origin introduced to the flock in adulthood became infected, demonstrating that lateral transmission had occurred. Lambs born to introduced ewes became infected and died at the same age as lambs born to native ewes, consistent with lateral transmission of scrapie to lambs. Although maternal transmission cannot be totally excluded for the lambs in this study, the data are consistent with lateral transmission being the most important means of spread leading to the high incidence of scrapie observed in this flock.
Research in Veterinary Science 07/2004; 76(3):211-7. · 1.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Major determinants of the pathological phenotype of natural scrapie are considered to be the agent strain and host prion protein (PrP) genotype, but the relationship between these is far from clear. Little is known about the strains that produce natural scrapie. A method of brain vacuolation profiling was developed which enables this aspect of disease phenotype to be characterized in detail. This method distinguished at least two distinct pathological phenotypes in sheep of a single genotype (ARQ/ARQ) from different flocks in the UK. Great similarity was also demonstrated between one of these phenotypes and the phenotype of sheep from a flock in Sardinia. The profile of four sheep of the same ARQ/ARQ genotype experimentally infected with bovine spongiform encephalopathy (BSE) was determined for comparison. It would appear from these preliminary observations that the application of lesion profiling techniques to ovine transmissible spongiform encephalopathy (TSE) may contribute to the definition of a particular scrapie phenotype within a flock. It may, therefore, have potential for improving our understanding of current TSE phenotypes in sheep, with regard to the possibility of identifying those of bovine origin.
Journal of Comparative Pathology 08/2002; 127(1):45-57. · 1.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Scrapie, bovine spongiform encephalopathy (BSE), and variant Creutzfeldt-Jakob disease belong to the group of disorders called transmissible spongiform encephalopathies or prion diseases. The possibility that some sheep may be infected with the BSE agent is of human and animal health concern. Immunohistochemical methods were used to identify specific prion protein (PrP) peptide sequences in specific cell types of the brain and lymphoreticular system (LRS) of sheep with natural scrapie and Suffolk and Romney sheep infected experimentally with the BSE agent. Clinically affected and some pre-clinical cases of BSE infection could be distinguished from scrapie cases by the lesser amount of labelling of PrP containing the 84-102 amino-acid peptide sequences in phagocytic cells of the LRS and brain. Additionally, BSE-infected sheep had higher degrees of intra-neuronal PrP accumulation in the brain, as detected by labelling for a range of PrP peptide sequences. These results suggest that there is strain-dependent processing of PrP in specific cell types within the nervous system and LRS which can be used to distinguish BSE- and scrapie-infected sheep.
Journal of Comparative Pathology 12/2001; 125(4):271-84. · 1.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sixty-three Romney sheep aged 6 months, consisting of three groups (PrP(ARQ/ARQ), PrP(ARQ/ARR), and PrP(ARR/ARR)genotypes) of 21 animals, were infected orally with brain tissue from BSE-infected cattle. Sub-groups of the 21 PrP(ARQ/ARQ) animals were killed, together with uninfected controls 4, 10, 16, 22 or 24-28 (after the development of full clinical disease) months post-inoculation (mpi). One sheep from each of the two groups of four killed at 4 or 10 mpi were shown by immunohistochemical examination to possess disease-specific PrP accumulations in single lymph nodes. At 16 mpi, such accumulations were detected in two of four infected sheep in some viscera and in the spinal cord and brain. At 22 mpi, three of five infected sheep had widespread disease-specific PrP accumulations in all tissues examined, but the remaining two animals gave positive results only in the central nervous system. Clinical disease appeared at 20-28 mpi. Three sheep killed with advanced clinical signs showed widespread PrP accumulation in brain, spinal cord and peripheral tissues. These results confirmed that PrP(ARQ/ARQ) Romney sheep are susceptible to experimental infection with the BSE agent. The different sites at which initial PrP accumulations were detected suggested that the point of entry of infection varied. Once established, however, infection appeared to spread rapidly throughout the lymphoreticular system. The results suggested that in some BSE-infected sheep neuroinvasion occurred in the absence of detectable PrP accumulations in the viscera or peripheral nervous system. In contrast to cattle with BSE, however, most sheep showed disease-specific PrP accumulations in the lymphoreticular system. In this respect, BSE-infected resembled scrapie-infected sheep; it is possible, however, that future research will reveal differences in respect of targeting of cell types within the lymphoreticular and peripheral nervous systems. The PrP(ARQ/ARR)and PrP(ARR/ARR)sheep were also killed in sub-groups at intervals after inoculation. Up to 24 mpi, however, none of these animals showed disease-specific PrP accumulations. Further results will be reported later.
Journal of Comparative Pathology 06/2001; 124(4):280-9. · 1.38 Impact Factor