Biology of PrPsc accumulation in two natural scrapie-infected sheep flocks.

Sheep scrapie is a prion disease that requires interaction of exogenous prions with host prion protein (PrP) supporting prion formation. Disease is associated with deposition of a host-generated conformational variant of PrP, PrPsc, in a variety of tissues, including brain, resulting in fatal spongiform encephalopathy. Efficiency of PrPsc formation is determined by polymorphisms in the PrP-coding sequence. This article adds to previous data of natural sheep scrapie, concentrating on the effect of host genotype and age on PrPsc accumulation patterns during preclinical and clinical disease. Two entire scrapie-infected, predominantly Suffolk-cross, sheep flocks euthanized for regulatory purposes were genotyped and analyzed for PrPsc deposition in various tissues using single- and dual-label immunohistochemistry. Scrapie, as defined by PrPsc deposition, occurred in 13/80 sheep. Preclinical disease was evident in nearly 70% of infected sheep, ranging in age from 14 months to 7 years. PrPsc accumulated systemically in the nervous tissue, various lymphoid tissues, both alimentary tract related and non-alimentary tract related, and the placenta. Clinical neurological illness was always associated with spongiform encephalopathy and PrPsc deposition in the brain. Only 6 of 9 sheep with preclinical scrapie had PrPsc deposition in the brain but widespread PrPsc deposition in peripheral lymphoid tissue, supporting previous data showing peripheral PrPsc accumulation preceding deposition in the brain. PrPsc colocalized with a marker for follicular dendritic cells throughout the lymphoid system. PrPsc also accumulated in the peripheral nervous system, particularly the nervous supply of the gastrointestinal tract. Abundant PrPsc was evident in trophoblast cells of placentomes but not in the endometrium, myometrium, or associated nervous plexus. PrPsc deposits were not observed in the mammary parenchyma or bone marrow. Scrapie susceptibility was defined genetically by PrP codon 171: PrPsc deposition was restricted to PrP genotype AA136RR154QQ171 in 12/13 cases or AV136RR154QQ171 in 1/13 cases. The earliest accumulation was observed in the single VRQ/ARQ heterozygous animal, consistent with the reported high scrapie susceptibility and brief incubation period observed in breeds with predominance of the V136R154Q171 allele. Disease occurred within, as well as independent of, mother-daughter lines, suggesting both maternal and nonmaternal transmission in the flocks.     

Age, scrapie status, PrP genotype and follicular dendritic cells in ovine ileal Peyer's patches

Follicular dendritic cells (FDCs) residing within ileal Peyer's patches (PPs) are of crucial relevance for sheep scrapie early pathogenesis and subsequent scrapie prion neuroinvasion. In this study, ileal PP follicles were significantly more numerous in lambs than in adult Sarda breed sheep, with significant differences being also found in lymphoid follicle area, perimeter and FDC density. Furthermore, PrPd deposition within ileal PPs and host's PrP genotype did not significantly influence these parameters. We conclude that age significantly affects FDC density in ileal PPs from Sarda breed ovines, independently from host's scrapie status and PrP genotype.     

Investigation of the effects of experimental autolysis on the detection of abnormal prion protein in lymphoid and central nervous system tissues from elk and sheep using the Western blotting method

Tissues unsuitable for standard immunohistochemical and histopathological examinations for chronic wasting disease (CWD) in cervids and for scrapie in sheep are frequently submitted for testing. This study investigated the effects of experimental autolysis on the detection of abnormal prion protein (PrPsc) in lymphoid and central nervous system (CNS) tissues from elk and sheep. The PrPsc was detected using a Western blotting (WB) test following PrPsc enrichment using sodium phosphotungstic acid (PTA) precipitation (PTA-WB). A commercial enzyme-linked immunosorbent assay (ELISA) was used as a reference test for quantitative measurement. This study showed that the amount of PrPsc in lymphoid and CNS tssues from elk and sheep decreased gradually as a result of autolysis, but PrPsc was still detectable after 5 and 15 d incubation at 37°C by PTA-WB for all lymphoid and CNS samples. The results of the ELISA supported those of PTA-WB, particularly for CNS tissues. In conclusion, autolysis at 37°C for 15 d would not significantly affect the detection of PrPsc in lymphoid and CNS tissues by WB and ELISA and, particularly, PTA-WB is a valuable and alternative confirmatory test to detect PrPsc in autolyzed lymphoid and CNS samples.     

Experimental transmission of abnormal prion protein (PrPsc) in the small intestinal epithelial cells of neonatal mice

Using an immunohistochemical method, we attempted to detect the transmission of abnormal prion protein (PrPsc) to the enterocytes of the small intestine of neonatal mice by oral exposure with sheep brain affected by scrapie. Five 1-day-old neonatal mice were exposed by oral inoculation to the homogenized brain of a scrapie-affected sheep. In the small intestine of all mice 1 hour after inoculation, immunoreactivity with antinormal prion protein (PrPc) antibody was seen in the cytoplasm of villus enterocytes. This finding suggests transmission of abnormal PrPsc into the cytoplasm of enterocytes. In control mice treated with normal sheep brain, no PrPc signal was seen in enterocytes of the small intestine. Immunopositivity for neurofilament protein and glial fibrillary acidic protein was seen in the cytoplasm of enterocytes of mice inoculated with scrapie and normal sheep brain. This suggests that the enterocytes of neonatal mice can absorb PrPsc and other macromolecular proteins of the sheep brain affected by scrapie and may be more important than previously thought as a pathway for PrPsc transmission in neonatal animals.     

Experimental transmission of scrapie agent to susceptible sheep by intralingual or intracerebral inoculation

Scrapie, a transmissible spongiform encephalopathy (TSE), is a naturally occurring fatal neurodegenerative disease of sheep and goats. This study documents survival periods, pathological findings, and the presence of abnormal prion protein (PrP(Sc)) in genetically susceptible sheep inoculated with scrapie agent. Suffolk lambs (AA/RR/QQ at codons 136, 154, and 171, respectively) aged 4 mo were injected by the intralingual (IL) or intracerebral (IC) route with an inoculum prepared from a pool of scrapie-affected US sheep brains. The animals were euthanized when advanced clinical signs of scrapie were observed. Spongiform lesions in the brain and PrPsc deposits in the central nervous system (CNS) and lymphoid tissues were detected by immunohistochemical and Western blot (WB) testing in all the sheep with clinical prion disease. The mean survival period was 18.3 mo for the sheep inoculated by the IL route and 17.6 mo for those inoculated by the IC route. Since the IC method is occasionally associated with anesthesia-induced complications, intracranial hematoma, and CNS infections, and the IL method is very efficient, it may be more humane to use the latter. However, before this method can be recommended for inoculation of TSE agents, research needs to show that other TSE agents can also transmit disease via the tongue.     

Features of follicular dendritic cells in ovine pharyngeal tonsil: an in vivo and in vitro study in the context of scrapie pathogenesis

Although the alimentary tract has been suggested as the most likely portal of entry in natural scrapie, a growing amount of data indicates that the respiratory system and more specifically the pharyngeal tonsils serve as a natural portal of entry for scrapie. This study describes for the first time the broad cell populations in the lymphoid compartment of pharyngeal tonsils and more specifically inside the lymphoid follicles where the scrapie agent accumulates during the period of latency. Follicular dendritic cells (FDCs), stromal cells located in the light zone of the germinal centre of lymphoid follicles, seem to be the principal causal factor in the accumulation of the infectious agent in transmissible spongiform encephalopathy (TSE) diseases. Knowing that efficient lymphoreticular prion propagation requires PrPc expression, we analysed the expression of PrPc with the mouse monoclonal antibody Pri 909 both in situ and on FDC-cluster-enriched cell suspensions. In situ, a positive staining was observed in the germinal centre of pharyngeal lymph follicles. The germinal centre labelling was due to the presence of a follicular dendritic network as revealed after immunogold staining of isolated FDC clusters. Our results suggest that the pharyngeal lymphoreticular system and more specifically PrPc expressing follicular dendritic cells could serve as a prion "reservoir" during the latency phase, thus playing a key role during the scrapie lymphoinvasion.     

White-tailed deer are susceptible to the agent of sheep scrapie by intracerebral inoculation

ABSTRACT: Interspecies transmission studies afford the opportunity to better understand the potential host range and origins of prion diseases. The purpose of this experiment was to determine susceptibility of white-tailed deer to the agent of scrapie after intracerebral inoculation and to compare clinical signs and lesions to those reported for chronic wasting disease (CWD). Deer (n = 5) were inoculated with 1 mL of a 10% (wt/vol) brain homogenate derived from a sheep clinically affected with scrapie. A non-inoculated deer was maintained as a negative control. Deer were observed daily for clinical signs of disease and euthanized and necropsied when unequivocal signs of scrapie were noted. One animal died 7 months post inoculation (pi) due to intercurrent disease. Examinations of brain tissue for the presence of the disease-associated abnormal prion protein (PrPSc) by western blot (WB) and immunohistochemistry (IHC) were negative whereas IHC of lymphoid tissues was positive. Deer necropsied at 15-22 months pi were positive for scrapie by IHC and WB. Deer necropsied after 20 months pi had clinical signs of depression and progressive weight loss. Tissues with PrPSc immunoreactivity included brain (at levels of cerebrum, hippocampus, colliculus, cerebellum, and brainstem), trigeminal ganglion, neurohypophysis, retina, spinal cord, and various lymphoid tissues including tonsil, retropharyngeal and mesenteric lymph nodes, Peyer's patches, and spleen. This work demonstrates for the first time that white-tailed deer are susceptible to sheep scrapie by intracerebral inoculation. To further test the susceptibility of white-tailed deer to scrapie these experiments will be repeated with a more natural route of inoculation.     

Immunohistochemical detection of scrapie prion proteins in clinically normal sheep in Pennsylvania.

Following diagnosis of scrapie in a clinically suspect Suffolk sheep, 7 clinically normal flockmates were purchased by the Pennsylvania Department of Agriculture to determine their scrapie status using an immunohistochemical procedure. Two of the 7 euthanized healthy sheep had positive immunohistochemical staining of the prion protein of scrapie (PrP-Sc) in their brains, nictitating membranes, and tonsils. The PrP-Sc was localized in the areas of the brain where, histopathologically, there was neurodegeneration and astrocytosis. The PrP-Sc occurred within germinal centers of the affected nictitating membranes and tonsils and was located in the cytoplasm of the dendrite-like cells, lymphoid cells, and macrophages. These results confirm that immunohistochemical examination of the nictitating membrane can be used as a screen for the presence of scrapie infection in clinically normal sheep at a capable veterinary diagnostic laboratory. In sheep with a PrP-Sc-positive nictitating membrane, the diagnosis of scrapie should be confirmed by histopathology and immunohistochemical examination of the brain following necropsy. Following full validation, immunohistochemistry assays for detection of PrP-Sc in nictitating membrane lymphoid tissues can improve the effectiveness of the scrapie control and eradication program by allowing diagnosis of the disease in sheep before the appearance of clinical signs.     

Immunohistochemical studies of scrapie archival material from Irish ARQ/ARQ sheep for evidence of bovine spongiform encephalopathy-derived disease

Since scrapie and bovine spongiform encephalopathy (BSE) in sheep are clinicopathologically indistinguishable, BSE in sheep may have been misdiagnosed as scrapie. Disease-specific prion protein (PrP(d)) patterns in archival tissues of 38 Irish ARQ/ARQ sheep diagnosed as scrapie-affected were compared to those in four Dutch BSE-challenged sheep. When medulla oblongata was immunolabelled with an antibody directed against amino acids 93-99 of ovine prion protein (ovPrP), intraneuronal PrP(d) was apparent in all 38 Irish sheep but was absent in BSE-challenged sheep. When lymphoid follicles were immunolabelled with antibodies directed against amino acids 93-106 of ovPrP, granule clusters of PrP(d) were seen in 34 of the 38 Irish sheep. Follicles of the remaining four archive sheep contained either no PrP(d) or single PrP(d) granules, similar to follicles from BSE-challenged sheep. Based on the medulla results, none of the archival cases had BSE-derived disease. The identification of some scrapie sheep with little or no intrafollicular PrP(d) suggests that this technique may be limited in discriminating between the two diseases.     

Immunohistochemical detection and distribution of prion protein in a goat with natural scrapie.

Formalin-fixed, paraffin-embedded tissue sections from a 3-year-old female Angora goat suffering from clinical scrapie were immunostained after hydrated autoclaving using a monoclonal antibody (mAb, F99/97.6.1; IgG1) specific for a conserved epitope on the prion protein. Widespread and prominent deposition of the scrapie isoform of the prion protein (PrPSc) was observed in the brain, brainstem, spinal cord, retina, postganglionic neurons associated with parasympathetic ganglia of myenteric and submucosal plexuses, Peyer's patches, peripheral lymph nodes, and pharyngeal and palatine tonsils. The goat was homozygous for PrP alleles encoding 5 octapeptide repeat sequences in the N-terminal region of the prion protein and isoleucine at codon 142, a genotype associated with high susceptibility and short incubation times in goats. The results of this study indicate that mAb F99/97.6.1 is useful for detection of PrPSc deposition, and this is a specific and reliable immunohistochemical adjunct to histopathology for diagnosis of natural caprine scrapie, although precise determination of the diagnostic sensitivity and specificity of the assay as a diagnostic test for scrapie in goats will require examination of a sufficiently large sample size. As with ovine scrapie, prion protein is widely distributed in the central and peripheral nervous systems, gastrointestinal tract, and lymphoid tissues in natural caprine scrapie.     

Experimental transmission of US scrapie agent by nasal, peritoneal, and conjunctival routes to genetically susceptible sheep

Scrapie is a naturally occurring fatal neurodegenerative disease of sheep and goats. This study documents incubation periods, pathologic findings, and distribution of abnormal prion proteins (PrP(Sc)) by immunohistochemistry in tissues of genetically susceptible sheep inoculated with US sheep scrapie agent. Four-month-old Suffolk lambs (QQ at codon 171) were inoculated by 1 of 3 different routes (nasal, peritoneal, and conjunctival) with an inoculum (No. 13-7) consisting of a pool of scrapie-affected sheep brains. Except for 3 sheep, all inoculated animals were euthanized when advanced clinical signs of scrapie were observed between 19 and 46 months postinoculation (MPI). Spongiform lesions in the brains and labeling of PrP(Sc) in central nervous system and lymphoid tissues were present in these sheep. One intranasally inoculated sheep euthanized at 12 MPI had presence of PrP(Sc) that was confined to the pharyngeal tonsil. These results indicate that the upper respiratory tract, specifically the pharyngeal tonsil, may serve as a portal of entry for prion protein in scrapie-infected environments.     

A histopathologic and immunohistochemical review of archived UK caprine scrapie cases

In 2005, a prion disease identified in a goat from France was reported to be consistent with disease from the bovine spongiform encephalopathy (BSE) agent. Subsequent retrospective examination of UK goat scrapie cases led to the identification of one potentially similar, but as yet unconfirmed, case from Scotland. These findings strengthened concerns that small ruminant populations exposed to the BSE agent have become infected. The lack of data relating specifically to scrapie in goats has been contributory to past assumptions that, in general, sheep and goats respond similarly to prion infections. In this study, brain material from 22 archived caprine scrapie cases from the UK was reviewed by histopathology and by immunohistochemical examination for accumulations of disease-specific prion protein (PrP(Sc)) to provide additional data on the lesions of caprine scrapie and to identify any BSE-like features. The vacuolar change observed in the goats was characteristic of transmissible spongiform encephalopathies in general. PrP(Sc) immunohistochemical morphologic forms described in scrapie and experimental BSE infections of sheep were demonstrable in the goats, but these were generally more extensive and variable in PrP(Sc) accumulation. None of the cases examined showed a PrP(Sc) immunohistochemical pattern indicative of BSE.     

Experimental transmission of sheep scrapie by intracerebral and oral routes to genetically susceptible Suffolk sheep in the United States.

Scrapie is a naturally occurring fatal neurodegenerative disease of sheep and goats. Susceptibility to the disease is partly dependent on the genetic makeup of the host. This study documents clinicopathological findings and the distribution of abnormal prion proteins (PrPres) by immunohistochemical and Western blot techniques, in tissues of genetically susceptible sheep inoculated with US sheep scrapie agents. Four-month-old Suffolk lambs (QQ or HQ at codon 171) were inoculated (5 intracerebrally and 19 orally) with an inoculum (#13-7) consisting of a pool of scrapie-affected sheep brains. Intracerebrally inoculated animals were euthanized when advanced clinical signs of scrapie were observed. Orally inoculated animals were euthanized at predetermined time points (4, 9, 12, 15, and 21 months postinoculation [PI]) and thereafter when the animals had terminal signs of disease. All intracerebrally inoculated animals exhibited clinical signs of scrapie and were euthanized between 13 and 24 months PI. Spongiform lesions in the brains and PrPres deposits in central nervous system and lymphoid tissues were present in these sheep. In orally inoculated sheep, clinical signs of scrapie were seen between 27 and 43 months PI in 5/9 animals. The earliest detectable PrPres was observed in brainstem and lymphoid tissues of a clinically normal, orally inoculated sheep at 15 months PI. Three of the 4 clinically normal sheep were positive at 15, 20, and 49 months PI by PrPres immunohistochemistry.     

我国部分地区传染性羊痒病基因型的分布情况调查

引起羊痒病的病原朊蛋白是一种正常的唾液酸糖蛋白(PrPc)在三级结构发生改变后形成的异常蛋白(PrPsc)。该病的发生与绵羊朊蛋白编码基因PRNP遗传多样性密切相关,主要表现在PRNP第136、154和171位密码子组成的PRNP基因型与绵羊对痒病的抗病性的联系。根据已建立的一种利用荧光定量PCR扩增反应对羊痒病抗性基因进行筛选的方法,对我国部分地区的无角多赛特绵羊羊痒病基因分布情况进行调查,从而从品种选育水平上杜绝羊痒病的发生。     

FDC-B1: a new monoclonal antibody directed against bovine follicular dendritic cells

Follicular dendritic cells (FDCs) are a unique population of accessory cells located in the light zone of the germinal centres of lymphoid follicles. Their involvement in the generation of humoral immune responses implies a potential role for these cells in many disorders. Indeed, in prion diseases, FDCs seem to be the major sites of extraneuronal cellular prion protein expression and the principal sites of the infectious agent accumulation in lymphoid organs. The identification of FDC is useful for the analysis of their distribution in reactive lymphoid tissue as well as in pathological conditions. The production and characterisation of a new mouse monoclonal antibody directed against bovine follicular dendritic cells (FDC-B1) is reported. The antigen detected by FDC-B1 is expressed exclusively on the surface of FDCs in ruminant lymphoid organs. The antigen has an approximate molecular weight of 28 kDa.     

Detection of prions in the faeces of sheep naturally infected with classical scrapie

ABSTRACT: Classical scrapie is a naturally transmitted prion disease of sheep and goats. Contaminated environments may contribute to the spread of disease and evidence from animal models has implicated urine, blood, saliva, placenta and faeces as possible sources of the infection. Here we sought to determine whether sheep naturally infected with classical scrapie shed prions in their faeces. We used serial protein misfolding cyclic amplification (sPMCA) along with two extraction methods to examine faeces from sheep during both the clinical and preclinical phases of the disease and showed amplification of PrPSc in 7 of 15 and 14 of 14 sheep respectively. However PrPSc was not amplified from the faeces of 25 sheep not exposed to scrapie. These data represent the first demonstration of prion shedding in faeces from a naturally infected host and thus a likely source of prion contamination in the environment.     

朊蛋白在宁夏滩羊不同组织中表达的免疫组化研究

作者运用免疫组织化学技术,首次对我国一类保护物种宁夏滩羊各组织中朊蛋白(PrP)的分布进行定性、定位研究。结果显示,PrP在滩羊的大脑、脑干、小脑、心脏、肝脏、脾脏、肾脏及淋巴结均有表达,尤其在脾脏、肾脏、脑干、淋巴结中的表达量丰富,而肺脏检测结果为阴性。此研究确定了朊蛋白在滩羊各组织中的表达与分布状况,并优化了朊蛋白检测的技术条件,为羊痒病的诊断与检测提供技术支持。     

Western blot detection of PrP Sc in archived paraffin-embedded brainstem from scrapie-affected sheep

Scrapie is a naturally occurring fatal neurodegenerative disease of adult sheep and goats, one of a group of mammalian diseases known as transmissible spongiform encephalopathies (TSE) or prion diseases. Immunoassays that identify disease-associated prion protein (PrP Sc) are integral to the diagnosis of scrapie and other prion diseases. Results obtained by either immunohistochemistry (IHC) or Western blot (WB) assay are generally adequate for the definitive diagnosis. Approved or accepted methods for WB diagnosis of TSEs requires the use of fresh or frozen nonfixed tissue samples, whereas formalin-fixed, paraffin-embedded tissue is required for the localization of PrP Sc by IHC. Because disparate processing methods are used for these accepted diagnostic techniques, separate tissue samples are collected from the same animal. Occasions arise in which there is either insufficient quantity of tissue available to complete analysis by both techniques or initial tissue processing is incompatible with one of the assays. Also, results between the assays may differ because of the vagaries of sampling, especially in case material that contains moderate-to-low levels of PrP Sc. The present article describes a method to conduct a WB assay from the same paraffin-embedded brainstem sample used for the IHC diagnosis of experimentally induced sheep scrapie.     

A case-control study on scrapie in Norwegian sheep flocks

Scrapie first was detected in indigenous sheep in Norway in 1981, and from 1995 to 1997 an increase in the number of flocks with scrapie cases was recorded. These flocks were mainly in one geographical region. A study to identify risk factors for scrapie was conducted. The study had three frequency-matched controls selected for every case within the same Veterinary District. A questionnaire was submitted to 176 sheep flocks (42 had been scrapie flocks). The data obtained by the questionnaire were linked to data collected from governmental and industry registers. After imputing missing data using single random imputation, the statistical analysis was performed using multivariable conditional logistic regression.

Purchase of female sheep from scrapie flocks, sharing of rams, or sharing of pastures between different flocks were the risk factors associated with the occurrence of scrapie. Of factors potentially sustaining and promoting the infection in the flock, number of winter-fed sheep, number of buildings for housing sheep, rams and ewes shared room during mating period and increase in the flock size were associated with scrapie. We interpret these findings to show that factors involving transfer of sheep between flocks or direct contact between sheep of different flocks are important for the spread of scrapie. Management factors are important for the development of scrapie. However, it was not possible to discriminate between the different management factors in this study at the flock level. Also, factors indicating awareness and interest of the farmer (as well as willingness to contact a veterinarian for diseased sheep) were related to the detection of scrapie in the flock.