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PubMed2000

Distribution of prion protein in the ileal Peyer's patch of scrapie-free lambs and lambs naturally and experimentally exposed to the scrapie agent.

A sensitive immunohistochemical procedure was used to investigate the presence of prion protein (PrP) in the ileal Peyer's patch of PrP-genotyped lambs, including scrapie-free lambs and lambs naturally and experimentally exposed to the scrapie agent. The tyramide signal amplification system was used to enhance the sensitivity of conventional immunohistochemical procedures to show that PrP was widely distributed in the enteric nervous plexus supplying the gut wall. In scrapie-free lambs, PrP was also detected in scattered cells in the lamina propria and in the dome and interfollicular areas of the Peyer's patch. In the follicles, staining for PrP was mainly confined to the capsule and cells associated with vascular structures in the light central zone. In lambs naturally exposed to the scrapie agent, staining was prominent in the dome and neck region of the follicles and was also found to be associated with the follicle-associated epithelium. Similar observations were made in lambs that had received a single oral dose of scrapie-infected brain material from sheep with a homologous and heterologous PrP genotype 1 and 5 weeks previously. These studies show that the ileal Peyer's patch in young sheep may be an important site of uptake of the scrapie agent and that the biology of this major gut-associated lymphoid tissue may influence the susceptibility to oral infection in sheep. Furthermore, these studies suggest that homology or heterology between PrP genotypes or the presence of PrP genotypes seldom associated with disease does not impede uptake of PrP.

Animals↗
PubMed2002

New Zealand sheep with scrapie-susceptible PrP genotypes succumb to experimental challenge with a sheep-passaged scrapie isolate (SSBP/1).

Scrapie does not occur in New Zealand (NZ), although PrP gene alleles associated with susceptibility to the disease are found at relatively high frequencies in NZ sheep. The hypothesis that scrapie is a genetic disease of sheep is thus unlikely to be true. To confirm that NZ sheep are actually susceptible to scrapie infection, NZ sheep of various PrP genotypes were challenged by subcutaneous inoculation with a sheep-passaged scrapie isolate (SSBP/1). Showing similar PrP genetics to that seen in UK sheep, all NZ sheep carrying the VRQ PrP allele developed clinical signs typical of scrapie, with characteristic neurodegenerative changes and PrP(Sc) evident on histopathological examination of their brains and lymphoid tissues. The incubation periods recorded in NZ sheep were generally shorter than those found in UK sheep. The results confirm that New Zealand sheep are as susceptible as their UK counterparts to experimental scrapie infection by subcutaneous inoculation.

Animals↗
PubMed1987

Antisera to scrapie-associated fibril protein and prion protein decorate scrapie-associated fibrils.

Scrapie-associated fibrils (SAF) are an infection-specific structure observed in the unconventional-agent diseases. Polyclonal antisera raised to scrapie proteins were used to test the antigenic relationship between purified fibrils and SAF isolated from non-protease-treated synaptosomal-mitochondrial preparations. The experimental design utilized fibrils from scrapie strain 263K-infected hamsters, scrapie strain 139A-infected mice, and scrapie strain ME7-infected mice. Preparations were examined by negative-stain immune electron microscopy and Western blot analysis of the polypeptides. Fibrils and polypeptides from each preparation reacted with a rabbit antiserum raised to each of the following: hamster 263K prion protein (PrP 27-30), hamster 263K SAF protein, and mouse ME7 SAF protein. Immune electron microscopy and Western blot analysis revealed similar antigenic relationships among the three scrapie antisera. Thus, fibrils and polypeptides can be considered to be the same in each preparation. No reactivity of the fibrils was observed with antisera raised to Alzheimer neurofibrillary tangles or a synthetic peptide of cerebrovascular amyloid. Thus, the fibrils observed in purified preparations share structural and antigenic similarities plus biochemically related peptides with SAF present in non-protease-treated preparations.

Animals↗
PubMed2006

Transportation of prion protein across the intestinal mucosa of scrapie-susceptible and scrapie-resistant sheep.

To determine the mechanisms of intestinal transport of infection, and early pathogenesis, of sheep scrapie, isolated gut-loops were inoculated to ensure that significant concentrations of scrapie agent would come into direct contact with the relevant ileal structures (epithelial, lymphoreticular, and nervous). Gut loops were inoculated with a scrapie brain pool homogenate or normal brain or sucrose solution. After surgery, animals were necropsied at time points ranging from 15 min to 1 month and at clinical end point. Inoculum-associated prion protein (PrP) was detected by immunohistochemistry in villous lacteals and in sub-mucosal lymphatics from 15 min to 3.5 h post-challenge. It was also detected in association with dendritic-like cells in the draining lymph nodes at up to 24 h post-challenge. Replication of infection, as demonstrated by the accumulation of disease-associated forms of PrP in Peyer's patches, was detected at 30 days and sheep developed clinical signs of scrapie at 18-22 months post-challenge. These results indicate discrepancies between the routes of transportation of PrP from the inoculum and sites of de novo-generated disease-associated PrP subsequent to scrapie agent replication. When samples of homogenized inoculum were incubated with alimentary tract fluids in vitro, only trace amounts of protease-resistant PrP could be detected by western blotting, suggesting that the majority of both normal and abnormal PrP within the inoculum is readily digested by alimentary fluids.

Animals↗
PubMed1989

Ovine scrapie: follow-up of sheep belonging to an endemic scrapie-infected flock.

There is a close similarity between the unconventional virus-induced ovine scrapie and human Creutzfeldt-Jakob disease. Since infection might be transmitted orally, the ovine production of an endemically scrapie-infected farm was studied. About 80% of the annual production are sold (50% as butcher-meat, 30% as breeding animals), and scrapie appears in 20% of the sheep kept on the farm. Was the same proportion of butcher-meat animals scrapie-infected? Since the scrapie agent has been detected in "clinically normal" lambs, the same problem occurs with breeding-sheep and "apparently healthy" animals: are they carriers of the pathogenic agent? Are they responsible for the spread of the ovine disease and/or of the human disease?

Animals↗
PubMed2002

Polymorphic distribution of the ovine prion protein (PrP) gene in scrapie-infected sheep flocks in which embryo transfer was used to circumvent the transmissions of scrapie.

The genetic sequence of the ovine prion protein (PrP) gene between codons 102 and 175 with emphasis on ovine PrP gene codons 136 and 171 was determined, and the polymorphic distribution of the ovine PrP gene in the scrapie-exposed Suffolk embryo donors and offspring from these donors that were transferred to scrapie-free recipient ewes was investigated in this study. The most common genotype was AA(136)QQ(171) (70% and 63% in the donor and offspring flocks, respectively), which is considered a high risk genotype in US Suffolk sheep. Although embryos were collected from scrapie-positive donors and many embryos had the high risk genotype, no scrapie occurred in the resulting offspring. Based upon the results of this study, we conclude that vertical transmission of scrapie can be circumvented using embryo transfer procedures even when the offspring have the high risk genotype.

Alleles↗
PubMed1987

Scrapie associated fibrils in the diagnosis of scrapie in sheep.

Previous research has consistently demonstrated by electron microscopy the presence of scrapie associated fibrils in brain extracts prepared from mice and hamsters with clinical signs of experimental scrapie. In the present study similar fibrils were seen in all the brain extracts prepared from 11 Cheviot or Suffolk sheep with natural or experimental scrapie that had been diagnosed clinically and confirmed neuropathologically. They were not found in the brain extracts of nine sheep that did not have scrapie and which included four that had been injected with infected material but did not develop the disease. The presence of such fibrils can therefore be used as an additional diagnostic criterion for natural scrapie in sheep.

Animals↗
PubMed1984

Preclinical changes in weight of scrapie-infected mice as a function of scrapie agent-mouse strain combination.

Several inbred strains of mice were injected with different scrapie agents and their total body weight was monitored throughout the incubation period. As a control, mice were injected with normal mouse brain homogenate. For most combinations of scrapie agent and mouse strain, weights during the preclinical phase were similar to or lower than the average weight of controls. For some combinations there was a significant increase in weight (compared to controls) during the latter part of the preclinical phase of disease. The effect was dependent on both agent and mouse strain, i.e., in some cases a mouse strain showed the increase with one scrapie agent but not another and some scrapie agents caused the increase in one inbred strain of mouse but not in another strain. The increase in weight was due to accumulations of fat rather than a generalized increase in weight of various organs. With one mouse strain (SJL), there was increased vacuolation seen in the hypothalamus of mice injected with scrapie agents that showed the increase in weight compared to the lesion intensity with an agent which did not cause the weight increase.

Adipose Tissue↗
PubMed1992

Polymorphisms of a scrapie-associated fibril protein (PrP) gene and their association with susceptibility to experimentally induced scrapie in Cheviot sheep in the United States.

The duration of the incubation period for scrapie, a fatal transmissible neurodegenerative disorder of sheep and goats, is mainly determined by the Sip gene, which has 2 alleles (sA--susceptible and pA--resistant). A diagnostic test is not available to detect scrapie in live animals. We analyzed genomic DNA extracted from frozen sheep brains collected from Cheviot sheep of the United States that had been inoculated with the SSBP/1 scrapie inoculum. Digestion of the DNA with EcoRI or HindIII followed by the addition of a scrapie-associated fibril protein (PrP)-specific marker probe, yielded fragments of 6.8 (e1) and 4.0 (e3) kb, or 5.0 (h1) and 3.4 (h2) kb, respectively. Fragments e1 and h2 were associated with the histopathologic diagnosis of scrapie, and fragments e3 and h1 were associated with survival. A valine/alanine polymorphism within the PrP coding region that resulted in a BspHI site was further used to determine the genotype of these Cheviot sheep. Digestion of polymerase chain reaction fragments with BspHI resulted in an undigested fragment b- (0.840 kb), digested fragments b+ (0.460 and 0.380 kb), or both types of fragments. Survival time of b+/b+ homozygous sheep was significantly (P < 0.01) shorter (218 +/- 26.0 days) than survival time for b-/b- sheep (> 700 days after inoculation). Results indicated that b+ and b- are markers for the Sip sA and pA alleles, respectively. The intermediate duration of the incubation period for heterozygous sheep (b+/b-; 342.9 +/- 25.3 days) indicated that the Sip sA allele is expressed codominantly to the Sip pA allele.

Animals↗
PubMedDate not supplied

Identification of scrapie prion protein-specific mRNA in scrapie-infected and uninfected brain.

To date no nucleic acid has been found in the purified infectious agent which causes the spongiform encephalopathy known as scrapie. In an attempt to identify a unique scrapie virus-associated messenger RNA in tissues of infected animals, we have synthesized an oligonucleotide probe complementary to the mRNA sequence corresponding to the amino-acid sequence of the prion protein, PrP27-30 (ref. 1). We report here that, with this probe, a complementary DNA clone representing PrP27-30 was obtained from scrapie-infected mouse brain; the DNA sequence of this clone could be translated into a protein that matches exactly the published sequence of PrP27-30. The cDNA clone hybridized to a single 2.4-2.5-kilobase (kb) mRNA from both normal and scrapie-infected brain. Thus, the PrP27-30 mRNA is not uniquely associated with scrapie infectivity, suggesting that PrP27-30 may be a normal component of mouse and hamster brain.

Amino Acid Sequence↗
PubMed1986

Partial copurification of scrapie-associated fibrils and scrapie infectivity.

The association between scrapie infectivity and scrapie-associated fibrils (SAF) during a partial purification procedure for infectivity was investigated. Scrapie infectivity and SAF can be separated from most membrane components by subcellular fractionation of infected mouse brain to obtain a synaptosomal fraction, followed by detergent treatment and density gradient centrifugation. After different detergent treatments, with either octyl glucoside or sodium N-lauroyl sarcosinate, SAF showed differing sedimentation characteristics but nevertheless cosedimented with scrapie infectivity in both cases. Copurification under two different conditions provides more evidence that SAF may be a form of the infectious agent of scrapie.

Animals↗
PubMedDate not supplied

The distribution of scrapie-associated fibrils in neural and non-neural tissues of advanced clinical cases of natural scrapie in sheep.

The distribution of scrapie-associated fibrils (SAFs) throughout four brain regions, the pituitary gland, along the whole length of the spinal cord and in the sciatic nerve was assessed in 10 sheep terminally affected by scrapie and in four control sheep. Tonsils, retropharyngeal, broncho-mediastinal and mesenteric lymph nodes, the distal ileum, proximal colon and spleen were also examined for fibrils in all 14 sheep. Fibrils were detected in all four brain regions and throughout the length of the spinal cord in nine of the scrapie affected sheep. SAFs were not detectable in any of the sciatic nerve samples tested. In one of the 10 clinically affected sheep only minimal lesions were found by histopathology and fibrils were detected only from the cerebrum and one spinal cord region (taken at the C1 C2 vertebrae). Fibrils were not detected in the tonsils or retropharyngeal lymph nodes but were detected in other non-neural tissues of some of the scrapie-affected sheep. These tissues included pituitary gland, broncho-mediastinal and mesenteric portal lymph nodes, distal ileum, proximal colon and spleen. Fibrils could not be detected in any of the tissues taken from the four control sheep.

Animals↗
PubMed1998

Gene expression in scrapie. Cloning of a new scrapie-responsive gene and the identification of increased levels of seven other mRNA transcripts.

To define genes associated with or responsible for the neurodegenerative changes observed in transmissible spongiform encephalopathies, we analyzed gene expression in scrapie-infected mouse brain using "mRNA differential display." The RNA transcripts of eight genes were increased 3-8-fold in the brains of scrapie-infected animals. Five of these genes have not previously been reported to exhibit increased expression in this disease: cathepsin S, the C1q B-chain of complement, apolipoprotein D, and two previously unidentified genes denominated scrapie-responsive gene (ScRG)-1 and ScRG-2, which are preferentially expressed in brain tissue. Increased expression of the three remaining genes, beta2 microglobulin, F4/80, and metallothionein II, has previously been reported to occur in experimental scrapie. Kinetic analysis revealed a concomitant increase in the levels of ScRG-1, cathepsin S, the C1q B-chain of complement, and beta2 microglobulin mRNA as well as glial fibrillary acidic protein and F4/80 transcripts, markers of astrocytosis and microglial activation, respectively. In contrast, the level of ScRG-2, apolipoprotein D, and metallothionein II mRNA was only increased at the terminal stage of the disease. ScRG-1 mRNA was found to be preferentially expressed in glial cells and to code for a short protein of 47 amino acids with a strong hydrophobic N-terminal region.

Amino Acid Sequence↗
PubMed1987

Detection of scrapie-associated fibrils (SAF) and SAF proteins from scrapie-affected sheep.

Scrapie-associated fibrils (SAF) were detected by negative-stain electron microscopy in the brains (by two different isolation procedures) and spleens of sheep naturally and experimentally infected with scrapie. Although the numbers of SAF varied from case to case, the yield of SAF from brains of naturally affected sheep was lower than that from experimentally affected sheep. SAF-specific, protease-resistant proteins (PrPs) were detected by silver staining and western blot analysis in most samples of brain from experimentally affected sheep. PrPs, however, could be detected in only a limited number of natural cases of sheep scrapie because of the lower yields of SAF. PrPs from sheep SAF appear biochemically and antigenically similar to PrPs from other species infected with unconventional agents. This study further establishes the unique association of SAF and PrPs with natural or experimentally induced scrapie in its natural host.

Animals↗
PubMed1988

Effects of the antiserum against a fraction enriched in scrapie-associated fibrils on the scrapie incubation period in mice.

An antiserum against a fraction enriched for scrapie-associated fibrils (SAF), was examined for its effects on scrapie incubation period by inoculating mice either intraperitoneally or intracerebrally with various dilutions of the serum mixed with scrapie-infected mouse brain homogenate. After intraperitoneal inoculation the mean time of the incubation period increased with increasing concentrations of the antiserum in a statistically significant fashion, when the serum dilutions were made with phosphate-buffered saline. After intracerebral inoculation, however, there were no statistically significant differences between the control group and any of the antiserum-groups. When the antiserum dilutions were made with pre-immune serum, the mice inoculated intraperitoneally also showed no significant differences between the two groups. These results indicate that the specific antibodies to SAF have no effect on the scrapie infectivity.

Animals↗
PubMed1993

Isolation of scrapie agent from the placenta of sheep with natural scrapie in Japan.

A five-month-pregnant Suffolk sheep histologically diagnosed as spontaneous scrapie was studied. Western blot analysis was performed with rabbit serum against the sheep scrapie-associated fibrils (SAF). In the proteinase K (pk)-treated parental brain and spleen samples, three major bands (15 K, 18 K, and 23 K) were detected. These major bands were not detected from the placenta. Infectious agents were isolated in mice from the brain samples but not from the placental homogenates. In another case of a three-month-pregnant Corriedale sheep without any clinical sign of, but histologically diagnosed as scrapie, was also studied in a similar approach. In the parental brain samples, three major bands (15 K, 18 K and 23 K) were detected. SAF protein was not detected in the parental spleen and placenta. No bands reactive with the antiserum were detected in any other samples from the fetal brain and spleen in both cases. However, infectious agents were isolated in mice from both brain and placental homogenates. Since the placenta is an important site of natural infection, it is worthwhile to study these tissues for the epidemiological study of scrapie infection.

Animals↗
PubMed1988

Molecular pathology of scrapie-associated fibril protein (PrP) in mouse brain affected by the ME7 strain of scrapie.

Scrapie-associated fibrils (SAF) are disease-specific structures found in extracts of the brains of animals affected with scrapie. These structures are pathological aggregates of a normal host protein (PrP). Abnormal post-translational modification of PrP has been suggested to explain its aberrant properties in scrapie-affected brains and although there is a form of PrP in SAF indistinguishable in size from the protein in uninfected brain, lower-molecular-mass variants of PrP are also found in SAF fractions. We report the characterisation of the multiple forms of PrP found in SAF fractions purified from mouse brain affected by the ME7 strain of scrapie. The quantitatively major forms of PrP in SAF prepared without the use of proteinase K have the amino-terminal sequence Lys-Lys-Arg-Pro-Lys-Pro-Gly-Gly-, identical to that predicted for the amino-terminus of normal mouse brain PrP. However N-terminal cleavage of some PrP does occur in vivo within a domain of repetitive sequences at sites similar to but distinct from those cut by proteinase K in vitro. This suggests the conformation of the protein in aggregates in vivo does not differ extensively from that in detergent-treated SAF in vitro. We conclude that the size diversity of PrP in SAF is only partly due to N-terminal proteolysis and is independent of the proteolysis that occurs if proteinase K is used in the purification of SAF. Apart from proteolytic changes in the structure of PrP, we found a novel, as yet unidentified, amino-acid derivative of the arginine residue at position 3 in mouse PrP, which may predispose PrP to form SAF.

Amino Acid Sequence↗
PubMed2002

Failure to detect abnormal prion protein and scrapie-associated fibrils 6 wk after intracerebral inoculation of genetically susceptible sheep with scrapie agent.

Detection of the scrapie-associated protease-resistant prion protein (PrPres) in sheep brains in the early phase after intracerebral inoculation of the scrapie agent has not been documented. Fourteen 4-mo-old, genetically susceptible lambs (QQ homozygous at codon 171 of the PrP gene) were obtained for this study. Twelve lambs were inoculated intracerebrally with a brain suspension from sheep naturally affected with scrapie, and 2 served as uninoculated controls. Two inoculated animals were euthanized at each of 6 times postinoculation (1 h to 6 wk), and their brains were collected for histopathological study, for detection of PrPres by the Western blot technique and an immunohistochemical (IHC) method, and for the detection of scrapie-associated fibrils (SAF) by negatively stained electron microscopy (EM). Microscopic lesions associated with introduction of the inoculum were seen in the brains of inoculated animals at all 6 times. However, both the Western blot and IHC techniques did not detect PrPres after the initial 3 d postinoculation, nor did EM detect SAF in any of the samples. From these findings, it is presumed that until host amplification has occurred, the concentration of PrPres in inoculum is insufficient for detection by currently available techniques.

Animals↗