Scrapie Pathology Research Articles

A computational framework for improving genetic variants identification from 5,061 sheep sequencing data

BackgroundPan-genomics is a recently emerging strategy that can be utilized to provide a more comprehensive characterization of genetic variation. Joint calling is routinely used to combine identified variants across multiple related samples. However, the improvement of variants identification using the mutual support information from multiple samples remains quite limited for population-scale genotyping.ResultsIn this study, we developed a computational framework for joint calling genetic variants from 5,061 sheep by incorporating the sequencing error and optimizing mutual support information from multiple samples' data. The variants were accurately identified from multiple samples by using four steps: (1) Probabilities of variants from two widely used algorithms, GATK and Freebayes, were calculated by Poisson model incorporating base sequencing error potential; (2) The variants with high mapping quality or consistently identified from at least two samples by GATK and Freebayes were used to construct the raw high-confidence identification (rHID) variants database; (3) The high confidence variants identified in single sample were ordered by probability value and controlled by false discovery rate (FDR) using rHID database; (4) To avoid the elimination of potentially true variants from rHID database, the variants that failed FDR were reexamined to rescued potential true variants and ensured high accurate identification variants. The results indicated that the percent of concordant SNPs and Indels from Freebayes and GATK after our new method were significantly improved 12%−32% compared with raw variants and advantageously found low frequency variants of individual sheep involved several traits including nipples number (GPC5), scrapie pathology (PAPSS2), seasonal reproduction and litter size (GRM1), coat color (RAB27A), and lentivirus susceptibility (TMEM154).ConclusionThe new method used the computational strategy to reduce the number of false positives, and simultaneously improve the identification of genetic variants. This strategy did not incur any extra cost by using any additional samples or sequencing data information and advantageously identified rare variants which can be important for practical applications of animal breeding.

Transcriptional profiling of peripheral lymphoid tissue reveals genes and networks linked to SSBP/1 scrapie pathology in sheep

Transmissible spongiform encephalopathies (TSEs) are slow and progressive neurodegenerative diseases of humans and animals. The major target organ for all TSEs is the brain but some TSE agents are associated with prior accumulation within the peripheral lymphoid system. Many studies have examined the effects of scrapie infection on the expression of central nervous system (CNS) genes, but this study examines the progression of scrapie pathology in the peripheral lymphoid system and how scrapie infection affects the transcriptome of the lymph nodes and spleen. Infection of sheep with SSBP/1 scrapie resulted in PrP Sc deposition in the draining prescapular lymph node (PSLN) by 25 days post infection (dpi) in VRQ/VRQ genotype sheep and 75 dpi in tonsils and spleen. Progression of PrP Sc deposition in VRQ/ARR animals was 25 dpi later in the PSLN and 250 dpi later in spleen. Microarray analysis of 75 dpi tissues from VRQ/VRQ sheep identified 52 genes in PSLN and 37 genes in spleen cells that showed significant difference ( P ≤ 0.05) between scrapie-infected and mock-infected animals. Transcriptional pathway analysis highlighted immunological disease, cell death and neurological disease as the biological pathways associated with scrapie pathogenesis in the peripheral lymphoid system. PrP Sc accumulation of lymphoid tissue resulted in the repression of genes linked to inflammation and oxidative stress, and the up-regulation of genes related to apoptosis.

Kuru likened to scrapie: the story remembered

In my letter to the editor of the Lancet of 5 September 1959, I pointed out the striking similarity of kuru and scrapie and suggested that kuru, like scrapie, might be a transmissible disease expressed after a long incubation period (Hadlow 1959). The unlikely linkage of these two diseases came about fortuitously while I was an employee of the United States Department of Agriculture studying the pathology of scrapie at the then Agricultural Research Council Field Station at Compton in Berkshire, England, beginning in March 1958. A year later parasitologist William Jellison, a friend and colleague from Rocky Mountain Laboratory, Hamilton, Montana, where I had worked before coming to England, visited my wife and me in Compton on the afternoon of 28 June 1959. He was returning from meetings in Eastern Europe. At dinner that evening he casually mentioned an exhibit he saw the previous day at the Wellcome Medical Museum in London. It had to do with a strange brain disease affecting the primitive people in Papua New Guinea. He thought I might like to see it owing to my interest in neuropathology. Five days later I saw the exhibit in London. It was on kuru, the strange brain disease, describing its clinical features and illustrating the neuropathological changes in its victims. Neuronal degeneration and intense astrocytosis likened kuru to scrapie. The likeness was made even more so by the single and multilocular vacuoles in the perikaryon of large neurons. From the start I was drawn to them, for they were so much like those in scrapie (Hadlow 1992). Although unusual in human neuropathology, in veterinary pathology such vacuolated neuronal cell bodies had long been identified almost solely with scrapie. Indeed, they had become the well-entrenched neurohistological hallmark of the disease and were (and still are) counted on in making its post-mortem diagnosis. Because spongiform change in the grey matter neuropil lacked prominence in both kuru and scrapie at the time, it was not part of the resemblance I saw in them. Then too, the topographic pattern of the degenerative changes in the brain has a certain sameness in the two diseases, with accent on the cerebellar cortex. So whatever else kuru and scrapie have in common—similar natural histories and general clinical features—the essential link between them was neurohistological, as tenuous as that seemed then and later (Hadlow 1995). The close kinship of kuru and scrapie, only surmised in 1959, was made more certain when the experimental transmission of kuru to chimpanzees was reported in 1966. It was indeed a welcome revelation. As it turned out kuru and scrapie are broadly similar transmissible neurological diseases. Two years earlier scrapie gave up its long-held exclusiveness as the only known transmissible degenerative disease of the central nervous system when a rare neurological disease of ranch mink was shown to be cast in the scrapie mould. But here was a human disease cast in that mould—the first one. The distinction had unforeseen implications, not least in helping bring together seemingly diverse neurological diseases of man and animals now commonly called the transmissible spongiform encephalopathies (Hadlow 1999). That my letter in the Lancet had a role, however small it may have been, in identifying other diseases belonging to this distinctive family, of course, pleases me. After all it was but the consequence of a chance observation. Still, knowing something about scrapie, especially its pathology, helped me to see the resemblance of the two diseases at first glance. I am sure that would not have happened as readily had I not learned a lot about scrapie in sheep and goats and looked at many of their brains microscopically during the previous year, for I came to Compton knowing little about the disease. Nevertheless, the overall likeness of kuru and scrapie is such that no doubt sooner or later someone would have become aware of it, though probably not as I did. These sentiments highlight the story of the kuru–scrapie likeness as I remember it.

Neuroanatomical distribution of abnormal prion protein in naturally occurring atypical scrapie cases in Great Britain

Scrapie belongs to a group of diseases known as the transmissible spongiform encephalopathies or prion diseases. Two different categories of naturally occurring scrapie have been identified: classical scrapie, which was first recorded around 1750, and atypical scrapie or 'Nor-98', which was first identified in Norway in 1998. The molecular characteristics of atypical scrapie have been well defined, but detailed descriptions of the neuropathological phenotype are rare since the majority of cases have been detected through active surveillance programmes where only brainstem and cerebellum are collected for statutory diagnosis. In order to characterise the neuropathology of naturally occurring atypical scrapie in sheep, we examined multiple brain levels from 15 whole brains from field cases of atypical scrapie, both clinical suspects and fallen stock, collected in Great Britain between 2004 and 2006. We found that the distribution of disease-associated prion protein (PrP(Sc)) and vacuolation in atypical scrapie cases are very different to both classical scrapie and experimental bovine spongiform encephalopathy in sheep. Immunolabelling for PrP(Sc) is mild and restricted at the obex and more intense and widespread rostrally, particularly in the cerebellum, substantia nigra, thalamus and basal nuclei. Intracellular immunolabelling types are not seen, but distinctive white matter immunolabelling is widespread. Vacuolation associated with PrP(Sc) deposits was not observed in the brainstem neuroanatomical areas commonly affected in classical scrapie and bovine spongiform encephalopathy, but was instead most prominent in the cerebellar cortex and neocortex. This is the largest comprehensive descriptive study of atypical scrapie pathology to date, and provides baseline data against which other natural or experimental cases can be compared. It also reinforces the current recommendation to collect cerebellum in addition to brainstem to enable confident confirmation of this distinct disease phenotype within surveillance programmes.

Increased expression of osteopontin in the brain with scrapie infection

The expression of osteopontin (OPN) was studied in the brains of mice with scrapie. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis showed that the expression of OPN protein and mRNA was increased significantly in the scrapie-infected brains compared to the controls. The increased expression of OPN protein was largely matched with the PrP Sc accumulation. Immunohistochemically, OPN was intensely immunostained in neurons of the midbrain at the time of scrapie infection initiation. Particularly, OPN immunostaining was noted in the reactive astrocytes and some microglia in the scrapie brains, while those cells were devoid of OPN immunoreactivity in control brains. Overall, these findings suggest that some neurons affected by PrP Sc at an early stage of scrapie transiently express OPN but subsequently succumb to cell death at a later stage of scrapie; astroglial cells after scrapie infection are activated to express OPN; and increased OPN expression in these cells may play an important role in the pathology of scrapie. The precise role of OPN in scrapie needs further study.

Post-natal knockout of prion protein alters hippocampal CA1 properties, but does not result in neurodegeneration.

Prion protein (PrP) plays a crucial role in prion disease, but its physiological function remains unclear. Mice with gene deletions restricted to the coding region of PrP have only minor phenotypic deficits, but are resistant to prion disease. We generated double transgenic mice using the Cre-loxP system to examine the effects of PrP depletion on neuronal survival and function in adult brain. Cre-mediated ablation of PrP in neurons occurred after 9 weeks. We found that the mice remained healthy without evidence of neurodegeneration or other histopathological changes for up to 15 months post-knockout. However, on neurophysiological evaluation, they showed significant reduction of afterhyperpolarization potentials (AHPs) in hippocampal CA1 cells, suggesting a direct role for PrP in the modulation of neuronal excitability. These data provide new insights into PrP function. Furthermore, they show that acute depletion of PrP does not affect neuronal survival in this model, ruling out loss of PrP function as a pathogenic mechanism in prion disease and validating therapeutic approaches targeting PrP.

Determination of the frequency and distribution of vascular and parenchymal amyloid with polyclonal and N-terminalspecific PrP antibodies in scrapie-affected sheep and mice

Brains from 17 histopathologically confirmed cases of scrapie, five of which had congophilic vascular amyloid, were stained immunohistochemically for prion protein (PrP) using a polyclonal antibody. Two clinically suspect but...

Changes in the localization of brain prion proteins during scrapie infection

Prion proteins (PrP) were localized in the brains of normal and scrapie-infected hamsters by immunohistochemistry and Western blotting. PrP monoclonal antibodies and monospecific anti-PrP peptide sera, which react with both the cellular (PrPC) and scrapie (PrPSc) isoforms of the prion protein, were used to locate PrP in tissue sections. In normal hamsters, PrPC was located primarily in nerve cell bodies throughout the CNS; whereas, in the terminal stages of scrapie, PrP immunoreactivity was shifted to the neuropil and was absent from most nerve cell bodies. Prion proteins were not uniformly dispersed throughout the gray matter of scrapie-infected hamster brains; rather, they were concentrated in those regions that exhibited spongiform degeneration and reactive astrogliosis. Since earlier studies showed that the level of PrPC remains constant during scrapie infection as measured in whole brain homogenates and no antibodies are presently available that can distinguish PrPC from PrPSc, we analyzed individual brain regions by Western blotting. Analysis of proteinase K-digested homogenates of dissected brain regions showed that most of the regional changes in PrP immunoreactivity that are seen during scrapie infection are due to the accumulation of PrPSc. These observations indicate that the tissue pathology of scrapie can be directly correlated with the accumulation of PrPSc in the neuropil, and they suggest that the synthesis and distribution of the prion protein has a central role in the pathogenesis of this disorder.

PrP-expressing tissue required for transfer of scrapie infectivity from spleen to brain.

Much available evidence points to a pathological isoform of the prion protein PrP being the infectious agent that causes transmissible spongiform encephalopathies, but the mechanisms controlling the neurotropism of prions are still unclear. We have previously shown that mice that do not express PrP (Prnp[o/o] mice) are resistant to infection by prions, and that if a Prnp(+/+) neurograft is introduced into such animals and these are infected intracerebrally with scrapie, the graft but not the surrounding tissue shows scrapie pathology. Here we show that PrP-expressing neurografts in Prnp(o/o) mice do not develop scrapie histopathology after intraperitoneal or intravenous inoculation with scrapie prions. Prion titres were undetectable in spleens of inoculated Prnp(o/o) mice, but were restored to wild-type levels upon reconstitution of the host lymphohaemopoietic system with PrP-expressing cells. Surprisingly, however, i.p. or i.v. inoculation failed to produce scrapie pathology in the neurografts of 27 out of 28 reconstituted animals, in contrast to intracerebral inoculation. We conclude that transfer of infectivity from the spleen to the central nervous system is crucially dependent on the expression of PrP in a tissue compartment that cannot be reconstituted by bone marrow transfer. Thus the requirement for the normal isoform of PrP in peripheral tissues represents a bottleneck for the spread of prions from peripheral sites to the central nervous system.

Normal host prion protein necessary for scrapie-induced neurotoxicity.

Accumulation of the prion protein PrPSc, a pathological and protease-resistant isoform of the normal host protein PrPC, is a feature of prion disease such as scrapie. It is still unknown whether scrapie pathology comes about by neurotoxicity of PrPSc, acute depletion of PrPC, or some other mechanism. Here we investigate this question by grafting neural tissue overexpressing PrPC into the brain of PrP-deficient mice which are scrapie-resistant and do not propagate infectivity. After intracerebral inoculation with scrapie prions, the grafts accumulated high levels of PrPSc and infectivity and developed the severe histopathological changes characteristic of scrapie. Moreover, substantial amounts of graft-derived PrPSc migrated into the host brain. Even 16 months after inoculation no pathological changes were seen in PrP-deficient tissue, not even in the immediate vicinity of the grafts. Therefore, in addition to being resistant to scrapie infection, brain tissue devoid of PrPC is not damaged by exogenous PrPSc.

Unsuspected early neuronal loss in scrapie-infected mice revealed by morphometric analysis.

This study was undertaken to determine to what extent neuronal loss is a feature of scrapie pathology, using an experimental model in which infectivity and subsequent vacuolar lesions are well characterized but in which neuronal loss has not been previously identified. Intraocular infection with ME7 scrapie directs infection through the major projections of the optic nerve, which include the dorsal lateral geniculate nucleus (dLGN) on the contralateral side to the infected eye. Infectivity can be detected in the dLGN at 77 days post-infection and vacuolar lesions are first seen around halfway through the incubation period of 240 days. Morphometric assessment of neuron number in the dLGN was made on gallocyanin stained semi-serial sections from 5 infected and 5 normal brain-injected controls at 4 fifty-day intervals during the incubation period, and on clinically terminal mice. The number of neurons in the dLGN of the infected mice decreased steadily from around 20,000 at 50 days post-infection to under 2,000 in the terminal group. The loss was delayed in the ipsilateral dLGN, although terminal counts were the same for both sides. The onset of neuronal loss was coincident with initial vacuolar changes, and neuronal numbers were inversely proportional to the severity of vacuolation. It is concluded that scrapie infection causes a progressive neuronal loss that can be identified some 30-80 days after infectivity can be detected in the dLGN, long before the onset of clinical disease.

The neuropathological phenotype in transgenic mice expressing different prion protein constructs.

Neuropathologic examination of transgenic (Tg) mice which express different prion protein (PrP) constructs is essential because spongiform (vacuolar) degeneration of neurons, the distribution of PrPSc and whether PrP amyloid plaques form are the phenotypes of prion diseases. In Tg models of experimental scrapie, it was found that all of the parameters that define prion isolates ('strains') can be manipulated by changing the structure of PrP. In those studies, further evidence that PrPSc causes scrapie neuropathology and determines scrapie incubation time was obtained. In addition, the distribution of PrPSc in the brain was unique for each prion isolate. The implications of these findings are first, that prion isolates target different neuron populations for synthesis of nascent pathogenic PrPSc and, secondly, that prion isolate diversity is determined by neurons. In Tg mice which express mutated PrP mimicking human prion protein gene mutations linked to familial prion diseases, the neuropathological changes have been faithfully reproduced. A new age-related, neuromuscular disorder has also been identified in uninfected Tg mice which overexpress wild-type PrPc. All of the findings with different PrP constructs plus the absence of scrapie pathology in PrP null mice are the strongest argument that the prion protein is the main etiologic and pathogenic factor of prion disorders.

Molecular biology and pathology of scrapie and the prion diseases of humans.

Scrapie and bovine spongiform encephalopathy of animals and Creutzfeldt-Jakob and Gerstmann-Sträussler-Scheinker diseases of humans are transmissible and genetic neurodegenerative diseases caused by prions. Infectious prion particles are composed largely, if not entirely, of an abnormal isoform of the prion protein which is encoded by a chromosomal gene. An as yet unidentified post-translational process converts the cellular prion protein into an abnormal isoform. Scrapie neuropathology, incubation times, and prion synthesis in transgenic mice are controlled by the prion protein gene. Point mutations in the prion protein genes of animals and humans are genetically linked to development of neurodegeneration. Transgenic mice expressing mutant prion proteins spontaneously develop neurologic dysfunction and spongiform neuropathology. Studies of prion diseases may advance investigations of other neurodegenerative disorders and of how neurons differentiate, function for decades and grow senescent.

Pathogenesis and Pathology of Scrapie after Stereotactic Injection of Strain 22L in Intact and Bisected Cerebella

The mechanisms involved in the spread of scrapie within the brain remain unclear. To examine this issue the 22L scrapie strain was injected in one side of the cerebellum of mice in which the cerebellum had been bisected prior to injection. Another group of animals received the same injection into intact cerebella, i.e. without prior bisection. We found that bisection of the cerebella delayed the spread of scrapie agent from the injection site to the contralateral side of the cerebellum and that the occurrence of vacuolization was not as extensive and was markedly delayed in the uninjected side compared to its occurrence after injection in the intact cerebellum. Replication of agent in an area preceded the development of vacuolization in that area by several weeks. There was marked loss of Purkinje cells on the injected side of bisected cerebella, with no loss seen on the uninjected side. The incubation period of scrapie disease in mice injected after cerebellar bisection was significantly longer than after the injection of intact cerebella. The results in this study suggest that the scrapie agent spreads along intact nerve cell tracts, probably by axonal transport.

Influence of stereotaxically injected scrapie on neurotransmitter systems of mouse cerebellum

The 22L strain of scrapie was injected stereotaxically into the cerebellum of C57BL/6J mice to determine its effect on several cerebellar neurotransmitter systems during the early clinical stages of the disease. In this model vacuolar lesions are restricted to the cerebellum with no evidence of vacuolization in other brain regions. Although lesions develop throughtout all cell layers of the cerebellum, they are most severe in the granule cell layer. Modest but significant (P < 0.01) reductions in cerebellar weight, glutamate decarboxylase activity, and in the affinity of the N 6-[adenine-2,8- 3H]cyclohexyladenosine binding sites, were observed in scrapie affected mice. The densities of the high- and low-affinity adenosine receptors were unaffected. Adenosine receptors in the cerebellum are highly localized to the axon terminals of the glutamatergic, GABA receptive granule cells. GABA, benzodiazepine, glutamate, and muscarinic cholinergic receptors were significantly altered. In addition, the high-affinity uptake of glutamate, and the activity of choline acetyltransferase were not significantly changed. GABA high-affinity uptake was slightly increased. Even though the granule cell layer of the cerebellum had undergone severe vacuolation, only modest neurotransmitter changes were apparent. Although these results suggest a tenous relationship between scrapie pathology and the integrity of neurotransmitter systems, it is possible that compensatory neurochemical changes in uncompromised neuronal populations may have masked potentially specific neurotransmitter effects.

Changes in the localization of brain prion proteins during scrapie infection.

Prion proteins (PrP) were localized in the brains of normal and scrapie-infected hamsters by immunohistochemistry and Western blotting. PrP monoclonal antibodies and monospecific anti-PrP peptide sera, which react with both the cellular (PrPC) and scrapie (PrPSc) isoforms of the prion protein, were used to locate PrP in tissue sections. In normal hamsters, PrPC was located primarily in nerve cell bodies throughout the CNS; whereas, in the terminal stages of scrapie, PrP immunoreactivity was shifted to the neuropil and was absent from most nerve cell bodies. Prion proteins were not uniformly dispersed throughout the gray matter of scrapie-infected hamster brains; rather, they were concentrated in those regions that exhibited spongiform degeneration and reactive astrogliosis. Since earlier studies showed that the level of PrPC remains constant during scrapie infection as measured in whole brain homogenates and no antibodies are presently available that can distinguish PrPC from PrPSc, we analyzed individual brain regions by Western blotting. Analysis of proteinase K-digested homogenates of dissected brain regions showed that most of the regional changes in PrP immunoreactivity that are seen during scrapie infection are due to the accumulation of PrPSc. These observations indicate that the tissue pathology of scrapie can be directly correlated with the accumulation of PrPSc in the neuropil, and they suggest that the synthesis and distribution of the prion protein has a central role in the pathogenesis of this disorder.

Prions: Novel Infectious Pathogens

Publisher Summary This chapter discusses the properties of the scrapie prion and its relationship to the infectious agents causing other apparently similar diseases. Six diseases are probably caused by prions, including scrapie, transmissible mink encephalopathy (TME), chronic wasting disease (CWD), kuru, Creutzfeldt–Jakob disease, and CJD, and Gerstmann–Straussler syndrome (GSS). Scrapie is a disease of sheep and goats. It is a neurological disorder characterized by pruritus, incoordination, and ataxia of gait. The disease is progressive and invariably fatal, as are all other prion diseases. A prolonged incubation period precedes the onset of clinical illness. The pathology of scrapie is confined to the central nervous system (CNS). Many countries have scrapie surveillance programs. Because sheep with scrapie cannot be treated, they are destroyed. The purification of the scrapie agent has been crucial for the studies defining the properties of the infectious particle. Several different purification procedures have been reported. One involved the copurification of the scrapie agent and microsomes. Another involved the isolation of a “membrane-free” fraction after prolonged ultracentrifugation. Multiple studies have shown that the scrapie agent in crude preparations is resistant to nuclease digestion and to UV irradiation.

Glycosidase histochemistry in normal and scrapie mice, rats, sheep and goats

Histochemical tests for two glycoside hydrolases, β-glucuronidase (GUR) and N-acetyl-β-D-glucosaminidase (GAM) were carried out on sections of brain and other tissues from mice experimentally infected with scrapie and from control mice. Increased activity of both enzymes was detected in the central nervous system of mice affected with scrapie, compared with low levels in normal mice. Neither enzyme was significantly altered in other tissues. Scrapie-inoculated mice in one series were killed at intervals between 30 and 120 days after inoculation and the development of increasing GAM activity in brain sections was followed. Abnormal activity was first detected 58 days after inoculation i.e. about 6 weeks before the onset of the clinical phase, and progressively increased to very strong positive reactions, easily discernible macroscopically, in the clinical phase. The location and distribution of abnormal enzyme activity followed consistent, although differing, patterns with both enzymes. Comparable tests in scrapie-affected and normal rats, sheep and goats showed that increased GAM activity could be detected in the rat, but not in the sheep or goat. The location of normal GUR in sheep and goats was different from that in mice and rats, being predominatly found in large neurones as well as paravascular. Both in scrapie sheep and in scrapie goats there was a qualitative difference in intraneuronal GUR, manifested as cells containing GUR droplets. Ways in which this histochemical dimension may add to fundamental concepts of the disease process and correlate with other facets of scrapie pathology are discussed.