OBJECTIVETo establish a stable PrP(Sc) panel from brain tissues of experimental hamsters infected with scrapie agent 263K for evaluating diagnostic techniques of human and animals' prion diseases.

METHODSThirty brain tissue samples from hamsters intracerebrally infected with scrapie strain 263K and another 30 samples from normal hamsters were selected to prepare 10%, 1%, and 0.5% brain homogenates, which were aliquoted into stocks. PrP(Sc) in each brain homogenate was determined by proteinase K digestions followed by Western blot assay and partially by immunohistochemistry. Stability and glycoforms of PrP(Sc) were repeatedly detected by PrP(Sc)-specific Western blots in half a year and 3 years later.

RESULTSPrP(Sc) signals were observed in all 10% brain homogenates of infected hamsters. Twenty out of 30 stocks and 19 out of 30 stocks were PrP(Sc) positive in 1% and 0.5% brain homogenatesof infected hamsters, respectively. Twenty-seven out of 30 stocks presented three positive bands in 10% brain homogenates, whereas none of 1% and 0.5% homogenates contained 3 bands. The detection of PrP(Sc)-specific signals stored in half a year and 3 years later demonstrated that the ratio of PrP(Sc) positive samples and glycoforms was almost unchanged. All normal hamsters' brain homogenates were PrP(Sc) negative.

CONCLUSIONA PrP(Sc) panel of prion disease can be established, which displays reliably stable PrP(Sc)-specific signals and glycoforms.

Animals ; Brain ; Cricetinae ; Immunohistochemistry ; Male ; PrPSc Proteins ; classification ; Scrapie

BACKGROUNDTo evaluate the influence ultrasonic processing on the aggregation of PrP(Sc) in the brain extracts prepared from the scrapie-infected hamsters, and to seek for the way to prepare lower molecular PrP(Sc) polymer.

METHODSThe extracts of infected brains were prepared with a lysis solution, and treated with ultrasonics at various conditions during the different phases. The distribution and aggregation state of PrP(Sc) were analyzed by proteinase K treated. Western blot, and afterwards, quantitatively calculated with a commercially supplied software Image Totaltech.

RESULTSThe amount of PrP(Sc) in the supernatant of brain homogenates was 1.29-to 1.58-fold increased with appropriate sonication (15 s for 30 times). At the same conditions of ultrasound, the PrP amount in the supernatant prepared from the scrapie-infected hamster brain was significantly increased, whereas that prepared from healthy animal used as normal control showed little change. Comparative analyses of PrP(Sc) pellets prepared by high-speed centrifugation revealed that about 90% PrP(Sc) released into supernatant after ultrasound processing.

CONCLUSIONAppropriate sonication of homogenate of scrapie-infected brain increases the extracted amount of PrP(Sc), being favorable to laboratory diagnosis. Larger molecular PrP(Sc) aggregates can be crashed by ultrasonic processing, engendering lower molecular PrP(Sc) polymers.

Animals ; Blotting, Western ; Brain Chemistry ; Cricetinae ; Image Processing, Computer-Assisted ; PrPSc Proteins ; analysis ; Scrapie ; metabolism ; Sonication

In sheep, susceptibility to scrapie is mainly determined by codons 136, 154, and 171 of the PRNP gene. Five haplotypes are usually present (ARR, ARQ, ARH, AHQ, and VRQ). The ARR haplotype confers the greatest resistance to classical scrapie while VRQ renders animals most susceptible. In 2004, the European Union implemented a breeding program that promotes selection of the ARR haplotype while reducing the incidence of VRQ. From 2006 to 2011 in Belgium, frequency for the ARR/ARR genotypes increased from 38.3% to 63.8% (n = 6,437), the ARQ haplotype diminished from 21.1% to 12.9%, and the VRQ haplotype decreased from 2.0% to 1.7%. The status of codon 141, a determinant for atypical scrapie, was also evaluated. Out of 27 different breeds (n = 5,163), nine were abundant. The ARR/ARR frequency increased in eight of these nine major breeds. The selection program has had a major impact on the ARR haplotype frequency in Belgium. However, the occurrence of atypical scrapie represents a critical point for this program that warrants the continuous monitoring of scrapie. Additionally, genotype frequencies among the breeds varied greatly. Texel, a breed that is common in Belgium, can still be selected for due to its average ARR frequency.
Animals ; Belgium ; *Breeding ; Female ; *Genetic Predisposition to Disease ; Genetic Variation ; *Genotype ; Male ; Scrapie/*genetics ; Sheep

OBJECTIVE: To describe the global profiles of acetylated proteins in the brains of scrapie agents 139A- and ME7-infected mice collected at mid-early, mid-late, and terminal stages. METHODS: The acetylated proteins from the cortex regions of scrapie agent (139A- and ME7)-infected mice collected at mid-early (80 days postinfection, dpi), mid-late (120 dpi), and terminal (180 dpi) stages were extracted, and the global profiles of brain acetylated proteins were assayed with proteomic mass spectrometry. The proteins in the infected mice showing 1.5-fold higher or lower levels than that of age-matched normal controls were considered as differentially expressed acetylated peptides (DEAPs). RESULTS: A total of 118, 42, and 51 DEAPs were found in the brains of 139A-80, 139A-120, and 139A-180 dpi mice, respectively. Meanwhile, 390, 227, and 75 DEAPs were detected in the brains of ME7-80, ME7-120, and ME7-180 dpi mice, respectively. The overwhelming majority of DEAPs in the mid-early stage were down-regulated, and more portions of DEAPs in the mid-late and late stages were up-regulated. Approximately 22.1% (328/1,485) of acetylated peptides mapped to 74 different proteins were mitochondrial associated. Kyoto Encyclopedia of Genes and Genomes pathway analysis identified 39 (80 dpi), 13 (120 dpi), and 10 (180 dpi) significantly changed pathways in 139A-infected mice. Meanwhile, 55, 25, and 18 significantly changed pathways were observed in the 80, 120, and 180 dpi samples of 139A- and ME7-infected mice ( P < 0.05), respectively. Six pathways were commonly involved in all tested samples. Moreover, many steps in the citrate cycle (tricarboxylic acid cycle) were affected, represented by down-regulated acetylation for relevant enzymes in the mid-early stage and up-regulated acetylation in the mid-late and late stages. CONCLUSION Our data here illustrated the changes in the global profiles for brain acetylated proteins during prion infection, showing remarkably inhibited acetylation in the early stage and relatively enhanced acetylation in the late stage.
Animals ; Brain/metabolism* ; Citrates/metabolism* ; Mice ; Peptides/metabolism* ; PrPSc Proteins ; Proteomics ; Scrapie/metabolism* ; Sheep

Scrapie is diagnosed antemortem in sheep by detecting misfolded isoforms of prion protein (PrP(Sc)) in lymphoid follicles of the rectal mucosa and nictitating membranes. Assay sensitivity is limited if (a) the biopsy is collected early during disease development, (b) an insufficient number of follicles is collected, or (c) peripheral accumulation of PrP(Sc) is reduced or delayed. A blood test would be convenient for mass live animal scrapie testing. Currently approved techniques, however, have their own detection limits. Novel detection methods may soon offer a non-animal-based, rapid platform with detection sensitivities that rival the prion bioassay. In anticipation, we sought to determine if diseased animals could be routinely identified with a bioassay using B lymphocytes isolated from blood sample volumes commonly collected for diagnostic purposes in small ruminants. Scrapie transmission was detected in five of six recipient lambs intravenously transfused with B lymphocytes isolated from 5~10 mL of blood from a naturally scrapie-infected sheep. Additionally, scrapie transmission was observed in 18 ovinized transgenic Tg338 mice intracerebrally inoculated with B lymphocytes isolated from 5~10 mL of blood from two naturally scrapie-infected sheep. Based on our findings, we anticipate that these blood sample volumes should be of diagnostic value.
Animals ; B-Lymphocytes/*pathology ; Biological Assay/*veterinary ; Mice ; Mice, Transgenic ; Prions/*blood ; Scrapie/blood/*diagnosis/transmission ; Sheep

OBJECTIVETo study the possible effect of tetracycline on protease-resistant activity in vitro and infectivity in vivo of a scrapie strain 263K.

METHODSScrapie pathogens were incubated with tetracycline at different concentrations for various periods of time and protease-resistant PrP signals were evaluated with proteinase K-treatment and Western blots. The preparations treated with tetracycline were intracerebrally inoculated into golden hamsters and typical TSE manifestations were noted. PrPSc in brain tissues of the infected animals was detected by PrP specific Western blot assays.

RESULTSProtease-resistant PrP was significantly reduced in or removed from the preparations treated with tetracycline in a dose-dependant manner. Compared with the control group after incubated for 53.75 +/- 0.50 days, the preparations treated with 5 mmol/L and 20 mmol/L tetracycline prolonged the incubation time of 61.5 +/- 1.73 and 59.5 +/- 0.58 days (P < 0.05).

CONCLUSIONTreatment of scrapie pathogen 263K with tetracycline reduces or removes its protease-resistant activity in vitro.

Animals ; Brain ; pathology ; Cricetinae ; Peptide Hydrolases ; metabolism ; PrPSc Proteins ; metabolism ; pathogenicity ; Scrapie ; pathology ; Tetracycline ; pharmacology ; Time Factors

Scrapie is a mammalian transmissible spongiform encephalopathy or prion disease that predominantly affects sheep and goats. Scrapie has been shown to overcome the species barrier via experimental infection of other rodents. To confirm the re-transmissibility of the mouse-adapted ME7 scrapie strain to ovine prion protein (PrP) transgenic mice, mice of an ovinized transgenic mouse line carrying the Suffolk sheep PrP gene that contained the A₁₃₆ R₁₅₄ Q₁₇₁/ARQ allele were intracerebrally inoculated with brain homogenates obtained from terminally ill ME7-infected C57BL/6J mice. Herein, we report that the mouse-adapted ME7 scrapie strain was successfully re-transmitted to the transgenic mice expressing ovine PrP. In addition, we observed changes in the incubation period, glycoform profile, and pattern of scrapie PrP (PrP(Sc)) deposition in the affected brains. PrP(Sc) deposition in the hippocampal region of the brain of 2nd-passaged ovine PrP transgenic mice was accompanied by plaque formation. These results reveal that the mouse-adapted ME7 scrapie strain has the capacity to act as a template for the conversion of ovine normal monomeric precursors into a pathogenic form in ovine PrP transgenic mice. The change in glycoform pattern and the deposition of plaques in the hippocampal region of the brain of the 2nd-passaged PrP transgenic mice are most likely cellular PrP species dependent rather than being ME7 scrapie strain encoded.
Alleles ; Animals ; Brain ; Gliosis ; Goats ; Humans ; Mice ; Mice, Transgenic ; Plaque, Amyloid ; Prion Diseases ; PrPSc Proteins ; Rodentia ; Scrapie ; Sheep ; Terminally Ill

Binding sites of five monoclonal antibodies were obtained by reinforceable method of overlapping recombinant prion protein and synthetic peptide. Overlapping peptides of PrP core were expressed in Escherichia coli by insertion of serial PCR amplicons of ovine PrP gene fragments into pET32a. The expressed fusion peptides were then tested for the binding activity to PrP monoclonal antibodies in Western blotting. The binding sites of 5 monoclonal antibodies of ovine PrP were located respectively as follows: 2H3 in 199 aa-213 aa, 4C6, 5F11 and 7F11 in 139 aa-168 aa and 7F1 in 214 aa-227 aa. There oligo peptides were synthesized and used in ELISA test for more accurate localization of the binding sites. The binding sites of 4C6, 5F11 and 7F11 were further confirmed to be in 149 aa-158 aa. This conclusion may contribute to the research for pathogenesis and diagnostic method of scrapie and bovine transmissible spongiform encephalopathy.
Animals ; Antibodies, Monoclonal ; immunology ; metabolism ; Binding Sites, Antibody ; immunology ; Epitopes ; immunology ; Escherichia coli ; genetics ; metabolism ; Prion Diseases ; diagnosis ; Prions ; genetics ; immunology ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Scrapie ; diagnosis ; Sheep

OBJECTIVETo understand the infectious characteristics of a hamster-adapted scrapie strain 263K with five different routes of infection including intracerebral (i.c.), intraperitoneal (i.p.), intragastrical (i.g.), intracardiac and intramuscular (i.m.) approaches.

METHODSHamsters were infected with crude- or fine-prepared brain extracts. The neuropathological changes, PrP(Sc) deposits, and patterns of PK-resistant PrP were analyzed by HE stain, immnunohistochemistry (IHC) assay and Western blot. Reactive gliosis and neuron loss were evaluated by glial fibrillary acidic protein (GFAP) and neuron specific enolase (NSE) specific IHC.

RESULTSThe animals inoculated in i.m. and i.p. ways with crude PrP(Sc) extracts showed clinical signs at the average incubation of 69.2 +/- 2.8 and 65.5 +/- 3.9 days. Inoculation in i.c. and intracardiac ways with fine PrP(Sc) extracts (0.00035 g) caused similar, but relative long incubation of around 90 days. Only one out of eight hamsters challenged in i.g. way with low dosage (0.01 g) became ill after a much longer incubation (185 d), while all animals (4/4) with high dosage (0.04 g) developed clinical signs 105 days postinfection. The most remarkable spongiform degeneration and PrP(Sc) deposits were found in brain stem among the five challenge groups generally. The number of GFAP-positive astrocytes increased distinctly in brain stems in all infection groups, while the number of NSE-positive cells decreased significantly in cerebrum, except i.c. group. The patterns of PK-resistant PrP in brains were basically identical among the five infection routes.

CONCLUSIONTypical TSE could be induced in hamsters by inoculating strain 263K in the five infection ways. The incubation periods in bioassays depend on infective dosage, administrating pathway and preparation of PrP(Sc). The neuropathological changes and PrP(Sc) deposits seem to be related with regions and inoculating pathways.

Administration, Oral ; Animals ; Blotting, Western ; Brain ; metabolism ; pathology ; Cricetinae ; Gliosis ; metabolism ; pathology ; Immunohistochemistry ; Injections, Intramuscular ; Injections, Intraperitoneal ; Injections, Intraventricular ; Neurons ; enzymology ; pathology ; Phosphopyruvate Hydratase ; metabolism ; Prions ; administration & dosage ; metabolism ; pathogenicity ; Scrapie ; metabolism ; pathology

OBJECTIVETo expatiate dynamic changes in hamsters infected with scrapie strain 263K, to observe the presence and aggravation of various forms of PrP and PrP(Sc) during incubation period, and to probe primarily the relationship between the onset of clinic manifestations and the presence of different PrP(Sc) forms.

METHODSHamster-adapted scrapie strain 263K was intracerebrally inoculated into hamsters. Different forms of PrP and PrP(Sc) were monitored dynamically by Western blot and immuno-histochemical assays. The presence of scrapie-associated fibril (SAF) was assayed by electron microscopy analysis (EM) and immuno-golden EM.

RESULTSPrP(Sc) was initially detected in the brain tissues of the animals in 20 days post-inoculation by immunohistochemistry and 40 days with Western blot. Quantitative evaluations revealed that the amounts of PrP and PrP(Sc) in brain tissues increased along with the incubation. Several high and low molecular masses of PrP were seen in the brains of the long-life span infected animals. Deglycosylation assays identified that the truncated PrP in the infected brains showed similar glycosylation patterns as the full-length PrP. The presence of short fragments was seemed to relate with the onset of clinical conditions.

CONCLUSIONThese results indicate that infectious agents exist and accumulate in central nerve system prior to the onset of the illness. Various molecular patterns of PrP(Sc) may indwell in brain tissues during the infection.

Animals ; Blotting, Western ; Brain ; metabolism ; ultrastructure ; Cricetinae ; Disease Models, Animal ; Female ; Glycosylation ; Immunohistochemistry ; Mesocricetus ; Microscopy, Electron ; PrP 27-30 Protein ; analysis ; metabolism ; PrPC Proteins ; analysis ; metabolism ; PrPSc Proteins ; analysis ; metabolism ; Scrapie ; metabolism ; pathology