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BMC Veterinary Research Jun 2006Diagnosis based on prion detection in lymph nodes of sheep and goats can improve active surveillance for scrapie and, if it were circulating, for bovine spongiform...
BACKGROUND
Diagnosis based on prion detection in lymph nodes of sheep and goats can improve active surveillance for scrapie and, if it were circulating, for bovine spongiform encephalopathy (BSE). With sizes that allow repetitive testing and a location that is easily accessible at slaughter, retropharyngeal lymph nodes (RLN) are considered suitable organs for testing. Western blotting (WB) of brain homogenates is, in principle, a technique well suited to both detect and discriminate between scrapie and BSE. In this report, WB is developed for rapid diagnosis in RLN and to study biochemical characteristics of PrPres.
RESULTS
Optimal PrPres detection in RLN by WB was achieved by proper tissue processing, antibody choice and inclusion of a step for PrPresconcentration. The analyses were performed on three different sheep sources. Firstly, in a study with preclinical scrapie cases, WB of RLN from infected sheep of VRQ/VRQ genotype--VRQ represents, respectively, polymorphic PrP amino acids 136, 154, and 171--allowed a diagnosis 14 mo earlier compared to WB of brain stem. Secondly, samples collected from sheep with confirmed scrapie in the course of passive and active surveillance programmes in the period 2002-2003 yielded positive results depending on genotype: all sheep with genotypes ARH/VRQ, VRQ/VRQ, and ARQ/VRQ scored positive for PrPres, but ARQ/ARQ and ARR/VRQ were not all positive. Thirdly, in an experimental BSE study, detection of PrPres in all 11 ARQ/ARQ sheep, including 7 preclinical cases, was possible. In all instances, WB and IHC were almost as sensitive. Moreover, BSE infection could be discriminated from scrapie infection by faster electrophoretic migration of the PrPres bands. Using dual antibody staining with selected monoclonal antibodies like 12B2 and L42, these differences in migration could be employed for an unequivocal differentiation between BSE and scrapie. With respect to glycosylation of PrPres, BSE cases exhibited a greater diglycosylated fraction than scrapie cases. Furthermore, a slight time dependent increase of diglycosylated PrPres was noted between individual sheep, which was remarkable in that it occurred in both scrapie and BSE study.
CONCLUSION
The present data indicate that, used in conjunction with testing in brain, WB of RLN can be a sensitive tool for improving surveillance of scrapie and BSE, allowing early detection of BSE and scrapie and thereby ensuring safer sheep and goat products.
Topics: Animals; Blotting, Western; Cattle; Encephalopathy, Bovine Spongiform; Lymph Nodes; Pharynx; PrPSc Proteins; Scrapie; Sheep
PubMed: 16764717
DOI: 10.1186/1746-6148-2-19 -
Virus Research Aug 2017Prion diseases are fatal neurodegenerative disorders by which the native cellular prion protein (PrP) is misfolded into an accumulating, disease-associated isoform...
Prion diseases are fatal neurodegenerative disorders by which the native cellular prion protein (PrP) is misfolded into an accumulating, disease-associated isoform (PrP). To improve the understanding of prion pathogenesis and develop effective treatments, it is essential to elucidate factors contributing to cellular permissiveness. We previously isolated five clones from an immortalized subline of ovine microglia, two of which had demonstrated differential permissiveness to a natural isolate of sheep scrapie and distinct transcriptomic profiles. To more robustly identify factors contributing to this activity, relative permissiveness, cell proliferation, selected gene transcript level, and matrix metalloproteinase 2 (MMP2) activity were compared amongst all five clones. Differences in cell proliferation were not detected between clones; however, significant correlations were identified between relative permissiveness and genes associated with cell growth (i.e., RARRES1 and PTN), protein degradation (i.e., CTSB and SQSTM1), and heparin binding (i.e., SEPP1). MMP2 activity varied amongst clones, but did not correlate with permissiveness. These associations support the contribution of cell division and protein degradation on the permissiveness of cultured ovine microglia to PrP.
Topics: Animals; Matrix Metalloproteinase 2; Microglia; PrPSc Proteins; Scrapie; Sheep; Transcriptome
PubMed: 28754560
DOI: 10.1016/j.virusres.2017.07.016 -
Virology Journal Mar 2012Transmissible spongiform encephalopathy (TSE) diseases are known to be zoonotic diseases that can infect different kinds of animals. The transmissibility of TSE, like...
BACKGROUND
Transmissible spongiform encephalopathy (TSE) diseases are known to be zoonotic diseases that can infect different kinds of animals. The transmissibility of TSE, like that of other infectious diseases, shows marked species barrier, either being unable to infect heterologous species or difficult to form transmission experimentally. The similarity of the amino acid sequences of PrP among species is believed to be one of the elements in controlling the transmission TSE interspecies. Other factors, such as prion strains and host's microenvironment, may also participate in the process.
METHODS
Two mouse-adapted strains 139A and ME7 were cerebrally inoculated to Golden hamsters. Presences of scrapie associate fibril (SAF) and PrPSc in brains of the infected animals were tested by TEM assays and Western blots dynamically during the incubation periods. The pathogenic features of the novel prions in hamsters, including electrophoretic patterns, glycosylating profiles, immunoreactivities, proteinase K-resistances and conformational stabilities were comparatively evaluated. TSE-related neuropathological changes were assayed by histological examinations.
RESULTS
After long incubation times, mouse-adapted agents 139A and ME7 induced experimental scrapie in hamsters, respectively, showing obvious spongiform degeneration and PrPSc deposits in brains, especially in cortex regions. SAF and PrPSc in brains were observed much earlier than the onset of clinical symptoms. The molecular characteristics of the newly-formed PrPSc in hamsters, 139A-ha and ME7-ha, were obviously distinct from the original mouse agents, however, greatly similar as that of a hamster-adapted scrapie strain 263 K. Although the incubation times and main disease signs of the hamsters of 139A-ha and ME7-ha were different, the pathogenic characteristics and neuropathological changes were highly similar.
CONCLUSIONS
This finding concludes that mouse-adapted agents 139A and ME7 change their pathogenic characteristics during the transmission to hamsters. The novel prions in hamsters' brains obtain new molecular properties with hamster-specificity.
Topics: Animals; Brain; Cricetinae; Endopeptidase K; Mesocricetus; Mice; Mice, Inbred C57BL; PrP 27-30 Protein; PrPSc Proteins; Protein Stability; Scrapie
PubMed: 22400710
DOI: 10.1186/1743-422X-9-63 -
Methods in Molecular Biology (Clifton,... 2017Protein misfolding cyclic amplification (PMCA) amplifies infectious prions in vitro. Over the past decade, PMCA has become an essential tool in prion research. The...
Protein misfolding cyclic amplification (PMCA) amplifies infectious prions in vitro. Over the past decade, PMCA has become an essential tool in prion research. The current chapter describes in detail the PMCA format with beads (PMCAb) and several methods that rely on PMCAb for assessing strain-specific prion amplification rates, for selective amplification of subtypes of PrP from a mixture, and a PMCAb approach that can replace animal titration of scrapie material. Development of PMCAb-based methodology is important for addressing a number of research topics including prion strain evolution, selection and adaptation, strain-typing, prion detection, and biochemical requirements for prion replication.
Topics: Animals; Biological Assay; Brain; Brain Chemistry; Cricetulus; Endopeptidase K; Gene Expression; Humans; Microspheres; PrPC Proteins; PrPSc Proteins; Protein Folding; RNA; Ribonuclease, Pancreatic; Scrapie; Sonication; Weaning
PubMed: 28861790
DOI: 10.1007/978-1-4939-7244-9_13 -
PLoS Pathogens Aug 2008The protease-resistant prion protein (PrP(res)) of a few natural scrapie isolates identified in sheep, reminiscent of the experimental isolate CH1641 derived from a...
The protease-resistant prion protein (PrP(res)) of a few natural scrapie isolates identified in sheep, reminiscent of the experimental isolate CH1641 derived from a British natural scrapie case, showed partial molecular similarities to ovine bovine spongiform encephalopathy (BSE). Recent discovery of an atypical form of BSE in cattle, L-type BSE or BASE, suggests that also this form of BSE might have been transmitted to sheep. We studied by Western blot the molecular features of PrP(res) in four "CH1641-like" natural scrapie isolates after transmission in an ovine transgenic model (TgOvPrP4), to see if "CH1641-like" isolates might be linked to L-type BSE. We found less diglycosylated PrP(res) than in classical BSE, but similar glycoform proportions and apparent molecular masses of the usual PrP(res) form (PrP(res) #1) to L-type BSE. However, the "CH1641-like" isolates differed from both L-type and classical BSE by an abundant, C-terminally cleaved PrP(res) product (PrP(res) #2) specifically recognised by a C-terminal antibody (SAF84). Differential immunoprecipitation of PrP(res) #1 and PrP(res) #2 resulted in enrichment in PrP(res) #2, and demonstrated the presence of mono- and diglycosylated PrP(res) products. PrP(res) #2 could not be obtained from several experimental scrapie sources (SSBP1, 79A, Chandler, C506M3) in TgOvPrP4 mice, but was identified in the 87V scrapie strain and, in lower and variable proportions, in 5 of 5 natural scrapie isolates with different molecular features to CH1641. PrP(res) #2 identification provides an additional method for the molecular discrimination of prion strains, and demonstrates differences between "CH1641-like" ovine scrapie and bovine L-type BSE transmitted in an ovine transgenic mouse model.
Topics: Animals; Antibodies, Monoclonal; Cattle; Disease Models, Animal; Encephalopathy, Bovine Spongiform; Female; Glycosylation; Mice; Mice, Transgenic; PrPSc Proteins; Protein Structure, Tertiary; Scrapie; Sheep; United Kingdom
PubMed: 18769714
DOI: 10.1371/journal.ppat.1000137 -
Journal of Virology Aug 2018Microglial cells in the central nervous system play important roles in neurodevelopment and resistance to infection, yet microglia can become neurotoxic under some...
Microglial cells in the central nervous system play important roles in neurodevelopment and resistance to infection, yet microglia can become neurotoxic under some conditions. An early event during prion infection is the activation of microglia and astrocytes in the brain prior to damage or death of neurons. Previous prion disease studies using two different strategies to manipulate signaling through the microglial receptor CSF-1R reported contrary effects on survival from prion disease. However, in these studies, reductions of microglial numbers and function were variable, thus confounding interpretation of the results. In the present work, we used oral treatment with a potent inhibitor of CSF-1R, PLX5622, to eliminate 78 to 90% of microglia from cortex early during the course of prion infection. Oral drug treatment early after infection with the RML scrapie strain significantly accelerated vacuolation, astrogliosis, and deposition of disease-associated prion protein. Furthermore, drug-treated mice had advanced clinical disease requiring euthanasia 31 days earlier than untreated control mice. Similarly, PLX5622 treatment during the preclinical phase at 80 days postinfection with RML scrapie also accelerated disease and resulted in euthanasia of mice 33 days earlier than infected controls. PLX5622 also accelerated clinical disease after infection with scrapie strains ME7 and 22L. Thus, microglia are critical in host defense during prion disease. The early accumulation of PrPSc in the absence of microglia suggested that microglia may function by clearing PrPSc, resulting in longer survival. Microglia contribute to many aspects of health and disease. When activated, microglia can be beneficial by repairing damage in the central nervous system (CNS) or they can turn harmful by becoming neurotoxic. In prion and prionlike diseases, the involvement of microglia in disease is unclear. Previous studies suggest that microglia can either speed up or slow down disease. In this study, we infected mice with prions and depleted microglia from the brains of mice using PLX5622, an effective CSF-1R tyrosine kinase inhibitor. Microglia were markedly reduced in brains, and prion disease was accelerated, so that mice needed to be euthanized 20 to 33 days earlier than infected control mice due to advanced clinical disease. Similar results occurred when mice were treated with PLX5622 at 80 days after infection, which was just prior to the start of clinical signs. Thus, microglia are important for removing prions, and the disease is faster when microglia are depleted.
Topics: Administration, Oral; Animals; Apoptosis; Disease Models, Animal; Female; Male; Mice; Microglia; Organic Chemicals; PrPSc Proteins; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; Scrapie; Severity of Illness Index
PubMed: 29769333
DOI: 10.1128/JVI.00549-18 -
The American Journal of Pathology Dec 2004Prion protein (PrP) is expressed in many tissues and is required for susceptibility to scrapie and other prion diseases. To investigate the role of PrP expression in... (Comparative Study)
Comparative Study
Prion protein (PrP) is expressed in many tissues and is required for susceptibility to scrapie and other prion diseases. To investigate the role of PrP expression in different cell types on pathology in retina and brain after scrapie infection, we examined transgenic mice expressing hamster PrP from the PrP promoter (tg7), the neuron-specific enolase promoter (tgNSE), or the astrocyte-specific glial fibrillary acidic protein promoter (tgGFAP). After intraocular inoculation with hamster scrapie, clinical disease developed in tg7 and tgNSE mice by 100 days and in tgGFAP mice by 350 days. Astrogliosis and scrapie-associated protease-resistant PrP (PrP-res) were detected in retina and brain before clinical onset. Retinal PrP-res was present in high amounts in both tg7 and tgNSE mice, however only tg7 mice developed retinal degeneration and extensive apoptosis. In contrast, in all three lines of mice high levels of brain PrP-res accompanied by neurodegeneration were observed. Thus, PrP expression on neurons or astrocytes was sufficient for development of scrapie-induced degeneration in brain but not in retina. The combined effects of PrP-res production in multiple cell types was required to produce retinal degeneration, whereas in brain PrP-res production by neurons or astrocytes alone was sufficient to cause neuronal damage via direct or indirect mechanisms.
Topics: Animals; Apoptosis; Astrocytes; Brain Diseases; Cricetinae; Disease Susceptibility; Eye; Green Fluorescent Proteins; In Situ Nick-End Labeling; Mice; Mice, Transgenic; Neurons; PrPSc Proteins; Retinal Degeneration; Scrapie
PubMed: 15579448
DOI: 10.1016/S0002-9440(10)63256-7 -
Journal of the Royal College of... 1994
Topics: Animals; Capsid; Cricetinae; Humans; Prion Diseases; Research; Scrapie
PubMed: 7965986
DOI: No ID Found -
Preventive Veterinary Medicine Nov 2018Certain genotypes of sheep have been identified to increase their susceptibility (the VRQ allele) or resistance (the ARR allele) to classical scrapie. This study's aim...
Certain genotypes of sheep have been identified to increase their susceptibility (the VRQ allele) or resistance (the ARR allele) to classical scrapie. This study's aim was to assess the spatio-temporal pattern of the ARR and VRQ alleles in Great Britain (GB) and to explore the risk factors associated to their presence. Data was collected from the GB scrapie active surveillance program, the sheep and goat inventory survey (GB census survey) and the agricultural survey for the period 2002-2015. Spatio-temporal trends of genotypes were assessed through the use of choropleth maps, spatial cluster and linear regression analyses. Multivariable mixed effect logistic regression analyses were performed to investigate the association between the resistant or susceptible genotypes, and breeds, farm purpose, animal purpose, surveillance stream, country location and herd size. The results show a significant upward trend in the frequency of most resistant ARR alleles (1.15% per year, 95%CI: 0.76-1.53) and significant downward trend of most susceptible VRQ alleles (-0.40% per year; 95%CI: -0.69 to -0.10]. The trend continues after the termination of the national scrapie plan in 2009. Breeds such as Herdwick (OR = 0,26; 95%CI: 0.14-0.46), Shetland (OR = 0.22; 95%CI: 0.13-0.39), Swaledale (OR = 0.58; 95%CI: 0.47-0.73), Scottish blackface (OR = 0.54; 95%CI: 0.41-0.71) and Welsh Montain (OR: 0.59; 95%CI: 0.44-0.79) were identified with lower odds ratios of having the resistant ARR allele, while Beulah speckled face (OR = 1.58; 95%CI: 1.04-2.41), Jacob (OR = 2.91; 95%CI: 1.33-6.40), Lleyn (OR = 2.94; 95%CI: 1.28-6.74) and Suffolk (OR = 2.19; 95%CI: 1.69-2.84) had higher odds ratios of having the ARR allele. Other risk factors associated to presence of ARR allele were finishing farms (OR = 1.15; 95%CI: 1.06-1.24) and farms in Scotland (OR = 0,78; 95%CI: 0.73-0.83) and in Lowland grazing areas (OR = 1.53; 95%CI: 1.39-1.67). Factors associated with presence the VRQ genotype were farms in Scotland (OR = 0,85; 95%CI: 0.77-0.93) and breeds such as Herdwick (OR = 2.2; 95%CI: 1.08-4.97), Shetland (OR = 4.12; 95%CI: 2.20-7.73) and Sweledale (OR = 1.51; 95%CI: 1.10-2.09). For the most resistant genotype, two significant spatial clusters were identified: a high-risk cluster in the south-west of GB (RR = 1.51, p < 0.001) and a low-risk cluster in northern GB (RR = 0.65, p < 0.001). For the most susceptible genotypes, one significant high-risk cluster was identified in Wales (RR = 2.89 and p = 0.013). Surveillance for classical scrapie could be improved with a risk-based approach by focussing on those areas and farm types identified to have higher frequency of VRQ alleles and less frequency of ARR alleles. Scrapie control strategies could focus on developing breeding programs on farms with Shetland, Herdwick and Swaledale breeds.
Topics: Alleles; Animals; Breeding; Disease Resistance; Genotype; Risk Factors; Scrapie; Sheep; Sheep Diseases; Sheep, Domestic; Spatio-Temporal Analysis; United Kingdom
PubMed: 30314774
DOI: 10.1016/j.prevetmed.2018.08.008 -
Comptes Rendus Biologies May 2002Despite experimental evidence that scrapie is an infectious disease of sheep, variations of the occurrence of the natural disease suggest an influence of host genetic... (Review)
Review
Despite experimental evidence that scrapie is an infectious disease of sheep, variations of the occurrence of the natural disease suggest an influence of host genetic factors. It has been established that the genetic polymorphism of the prion protein (PrP) gene is correlated to the incidence of scrapie and to the survival time: five polymorphisms have been described by variations at amino-acid codons 136, 154 and 171. In this paper we study the effect on scrapie susceptibility of the pairing of the five allelic variants known to exist: we show that scrapie susceptibility is given by the produce of the elementary allelic factors. This first well-documented evidence of a multiplicative property of genetic risk factors could give hints on the underlying mechanisms of prion-induced neurodegenerative diseases.
Topics: Alleles; Animals; Genetic Predisposition to Disease; Genetic Variation; Genotype; Prions; Scrapie; Sheep
PubMed: 12187642
DOI: 10.1016/s1631-0691(02)01465-8