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1.
The usefulness of detecting the scrapie-associated fibrillar protein (PrP) in the lymphoreticular organs of sheep as a diagnostic tool was investigated. The PrP was detected by means of a rabbit-anti-sheep PrP polyclonal antibody by Western blot analysis. PrP was detected in samples from the central nervous system (CNS) of five of six sheep showing clinical signs of natural scrapie infection, in spleen samples from four of the six sheep and in lymph node samples taken from three of the sheep. PrP was detected in the spleen and lymph node samples, but not in the CNS samples from one of the six sheep that was clinically and histopathologically abnormal. This animal appeared to be in the early clinical stage of the disease. A total of 47 clinically normal sheep were examined for the presence of PrP. It was detected in spleen samples from three of the 47 sheep and in lymph node samples from three of the 39 sheep tested. Similarly, PrP was detected in a sample of lymph node obtained surgically from one of three experimentally infected sheep 14 months after inoculation. The PrP-positive sheep and one of the remaining PrP-negative sheep showed clinical signs of scrapie six and five months later respectively. One sheep euthanased 18 months after experimental infection was positive for PrP in the CNS, spleen and lymph node, but five other sheep which were killed or died two, eight, 16, 18 and 21 months after infection were negative or doubtful for the detection of PrP.  相似文献   

2.
Natural scrapie, new variant Creutzfeldt-Jakob disease and murine experimental transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative disorders. The agent responsible for these diseases is closely related to PrPsc, an abnormal isoform of the cellular prion protein. Before reaching the brain, it invades and replicates in lymphoid organs such as spleen, tonsils and lymph nodes. Follicular dendritic cells (FDC) may support the prion replication in lymphoid tissues of sheep as shown in murine models infected with scrapie. In sheep, specific antibodies recognising FDC are lacking. The CNA42 mAb, directed against human FDC was used to identify these cells in sheep spleen. As well as showing that the pre-treatments needed for immunohistochemical detection of PrPsc did not prevent labelling by the CNA42 mAb, accumulation of PrPsc in FDC of spleens of scrapie affected sheep was demonstrated using a double immunolabelling strategy. Thus, the CNA42 antibody represents a suitable tool to identify FDC and investigate their role in natural sheep scrapie.  相似文献   

3.
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.  相似文献   

4.
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。  相似文献   

5.
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.  相似文献   

6.
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7.
Scrapie is a naturally occurring transmissible encephalopathy of sheep and goats. Currently available methods for diagnosis are the presence of characteristic histopathologic changes and detection of an abnormal form of prion protein (PrPres) in the brains of affected animals. This study documents preclinical and subclinical scrapie in a flock of 16 sheep utilizing histopathology, immunohistochemistry (IHC), western blot, and electron microscopy (for scrapie-associated fibrils) for confirmation of the disease. Prior to necropsy, none of the sheep showed signs of clinical scrapie. Based on the results of histopathology and positive PrPres tests, 3 ewes were found to have subclinical scrapie. An additional ewe, which did not have histopathologic changes in the brain but was positive by IHC and western blot,was considered a preclinical case of scrapie. None of the sheep had amyloid in the brain stem.  相似文献   

8.
Seventeen clinically suspect scrapie sheep, and twelve suspected BSE-affected cattle were confirmed using routine histopathological examination by the detection of characteristic spongiform change in the medulla brain region taken at the level of the obex. Three sheep and four cows acquired as controls showed no spongiform change. Five aliquots of brain tissue from each of four brain regions were taken (cerebellum, medulla, frontal cerebral cortex and occipital cerebral cortex) from each of the 36 animals. One aliquot was frozen at -70 degrees C, the others were subjected to one of four autolysis regimes at 3 or 7 days at 25 degrees C or 37 degrees C. All samples were tested by Western immunoblotting for detection of PrP(Sc) using the Prionics - Check test (Prionics AG, Zurich, Switzerland). Further samples of medulla from 15 suspect scrapie cases, 10 healthy sheep, 13 suspect BSE cows and 5 healthy cows, were taken adjacent to the obex, and subjected to autolysis at 37 degrees C for 6, 12, 24 and 48 hours before being fixed in 10 per cent formal saline and subsequently examined by a routine immunohistochemical technique for detection of PrP(Sc) protein. The abnormal protein could not be detected in any of the control animals by either technique. PrP(Sc) could be detected by Western immunoblotting in at least one brain area from all the positive animals after autolysis for 7 days at 37 degrees C. The protein could be detected by immunohistochemistry in all cases which were positive by histopathological examination using all autolysis conditions. From the results of this study it is concluded that autolysis does not significantly compromise the diagnosis of scrapie or BSE by either of these diagnostic methods.  相似文献   

9.
Meadow voles (Microtus pennsylvanicus) are permissive to chronic wasting disease (CWD) infection, but their susceptibility to other transmissible spongiform encephalopathies (TSEs) is poorly characterized. In this initial study, we intracerebrally challenged 6 meadow voles with 2 isolates of sheep scrapie. Three meadow voles acquired a TSE after the scrapie challenge and an extended incubation period. The glycoform profile of proteinase K-resistant prion protein (PrPres) in scrapie-sick voles remained similar to the sheep inocula, but differed from that of voles clinically affected by CWD. Vacuolization patterns and disease-associated prion protein (PrPSc) deposition were generally similar in all scrapie-affected voles, except in the hippocampus, where PrPSc staining varied markedly among the animals. Our results demonstrate that meadow voles can acquire a TSE after intracerebral scrapie challenge and that this species could therefore prove useful for characterizing scrapie isolates.  相似文献   

10.
A randomised sample of 2,809 apparently healthy sheep, 55 per cent of them less than 15 months of age, which were slaughtered for human consumption at abattoirs in Great Britain in 1997/98, was taken to establish the prevalence of scrapie infection. The medulla oblongata of each sheep was examined histopathologically at the level of the obex, and fresh brain tissue was examined for scrapie-associated fibrils (SAF) to establish whether there was evidence of scrapie. In addition, histological sections of the medulla from 500 of the sheep were immunostained with an antiserum to PrP, and the same technique was also applied to any animal found positive or inconclusive by the histological or SAF examinations. Any sheep which was positive by any of these diagnostic methods was also examined by Western immunoblotting, for the detection of the disease-specific protein PrP(Sc). A total of 2,798 sheep (99.6 per cent) were negative by all the methods applied. Ten animals were SAF-positive but negative by all the other methods, and in one animal there was immunohistochemical staining which could not be interpreted unequivocally as disease-specific. A mathematical model was used to estimate the prevalence of scrapie infection in the national slaughtered sheep population which would be consistent with these results. By this model, the absence of unequivocally substantiated cases of scrapie in the sample was consistent with a prevalence of infection in the slaughter population of up to 11 per cent.  相似文献   

11.
Scrapie associated fibrils in the diagnosis of scrapie in sheep   总被引：2，自引：0，他引：2  
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.  相似文献   

12.
One of the "gold standard" techniques for postmortem confirmation of scrapie diagnosis in sheep and goats is immunohistochemical examination of brain tissue. Active surveillance for scrapie is mainly performed by rapid diagnostic tests on the basis of postmortem immunochemical detection of prion protein (PrP) in the obex tissue. The aim of this study was to determine the performance of 2 rapid tests, Prionics-Check LIA (a chemiluminescence sandwich enzyme-linked immunosorbent assay) and Prionics-Check Western blot for scrapie diagnosis when applied to brain areas other than the obex, in comparison with the recognized immunohistochemistry. Prion protein was detected in the obex, cervical spinal cord, and thalamus from all the scrapie-positive sheep by the 3 tests. Western blot and LIA were negative in other areas of the brain, although weak immunohistochemical staining was detected. The results show that the 2 rapid tests studied may detect PrP in brain areas other than the obex, although with a lower sensitivity than immunohistochemistry when there is minimal PrP deposition.  相似文献   

13.
Summary

The present article (part I) reviews recent developments in animal spongiform encephalopathies (SEs), with the exception of bovine spongiform encephalopathy (BSE), which is dealt with in part II.

The article focuses on scrapie and describes epidemiological aspects and the prospects for a preclinical diagnosis. Up to now, confirmatory diagnosis of scrapie depended on histological examination of the brain, collected during post‐mortem examination from sheep with clinical signs of the disease. An altered protein, PrPSc, can be detected in the brain of diseased animals. The demonstration of the same protein in the spleen and in peripheral lymph nodes of infected animals seems to offer interesting possibilities of arriving at a method for a preclinical diagnosis, and thus a diagnosis in the live animal. Progress has also been made in our understanding of the relationship between the genetic constitution and susceptibility of the host. Susceptibility is expressed as the survival time of sheep inoculated with scrapie. This was thought to be determined by a single genetic locus designated the Sip gene (scrapie incubation period gene). Putative markers for the two alleles of the Sip gene, sA and pA, have been discovered, consisting of restriction fragment length polymorphisms (RFLPs). In field tests, however, the link between these markers and the length of incubation time was far from consistent. These RFLPs were found to be situated outside the prion‐protein‐co‐ding region of the ovine gene. In later studies, RFLPs were detected inside this region. These markers appear to be more informative, i.e. they correspond with a difference in the length of the scrapie incubation period.

Finally, the article briefly describes recent developments in other, lesser known, animal spongiform encephalopathies: chronic wasting disease and other spongiform encephalopathies in exotic ungulates, transmissible mink encephalopathy, and feline spongiform encephalopathy, focusing on their possible links with scrapie or bovine spongiform encephalopathy.  相似文献   

14.
To determine the levels of background scrapie-like pathology in the brains of clinically normal adult sheep, the brains of 1106 sheep from 28 known scrapie-infected flocks and nine apparently uninfected flocks were examined during 1998 and 1999. One per cent of the brains had vacuolar pathology and disease-specific accumulations of prion protein consistent with a diagnosis of scrapie. All the positive animals had at least one allele of the prion protein gene encoding valine at codon 136, and originated from flocks in which cases of clinical scrapie had been confirmed within the last four years. The parasympathetic nucleus of the vagal nerve was the most consistently and severely affected nucleus in the medulla oblongata, suggesting that the infection enters the brain via ascending fibres of the vagus nerve.  相似文献   

15.
The purpose of this study was to enhance the sensitivity of the Western blot (WB) test for use as an alternative and confirmatory method for the diagnosis of scrapie and chronic wasting disease (CWD) in Canada by comparing 2 sample preparation procedures: an abnormal prion protein (PrPSc) concentration procedure using sodium phosphotungstic acid (PTA) precipitation and a procedure using crude sample without precipitation. A total of 100 cerebrum samples (52 sheep and 48 elk), including 66 negative (31 sheep, 35 elk) and 34 positive (21 scrapie and 13 CWD positive) samples diagnosed by using immunohistochemistry (IHC) on retropharyngeal lymph node (RPLN) and medulla oblongata at obex, were tested by using WB with the 2 sample preparation procedures. The WB using non-PTA enriched sample (crude extract) detected, on average, only 71.7% (9 of 15, 60.0% for scrapie, 5 of 6, 83.3% for CWD) of the samples that tested positive by using WB with PTA enriched samples. No case was positive by WB using crude extract but negative by WB using PTA enriched sample. No false positive was found. Serial dilution of PTA precipitated samples demonstrated that the technique increases the detection limit approximately 100 fold. Additionally, the comparison of the WB and IHC on cerebrum from all the positive cases demonstrated that WB following PTA precipitation and IHC had 100% agreement by detecting 6 positive for CWD on cerebrum; while IHC detected scrapie in only 14 out of 15 positive cerebrum samples by using WB following PTA precipitation. Phosphotungstic acid precipitation is therefore a useful adjunct to WB analysis of scrapie and CWD and tissues.  相似文献   

16.
12只雌性5周龄金仓鼠腹腔注射50μL羊痒病(SCRAPIE)263K株10%的脑组织匀浆。在临床前期,感染后第2、4、6、8、10、12周和临床末期第20周,金仓鼠被分别处死,取大脑、淋巴组织、脾脏、肾脏、肝脏、心脏和肌肉等组织,进行蛋白质免疫印迹杂交实验检测朊病毒病理型朊蛋白PrPSc。研究结果显示在临床前期,通过改进后的纯化技术,我们能够检测出淋巴组织和脾脏组织中的PrPSc,但是在肾脏、肝脏、心脏和肌肉等组织中仍然没有检测到PrPSc。  相似文献   

17.
To obtain a more detailed understanding of the prevalence of classical scrapie infections in a heavily affected German sheep flock (composed of 603 sheep and 6 goats), we analysed 169 sheep and 6 goats that carried the genotypes susceptible to the disease and that were therefore culled following discovery of the index case. The initial tests were performed using the Biorad TeSeE ELISA and reactive results were verified by official confirmatory methods (OIE-immunoblot and/or immunohistochemistry (IHC)) to demonstrate the deposition of scrapie-associated PrP(Sc) in the brain stem (obex). This approach led to the discovery of 40 additional subclinically scrapie-infected sheep. Furthermore, peripheral lymphatic and nervous tissue samples of the 129 sheep and 6 goats with a negative CNS result were examined by IHC in order to identify any preclinical infections which had not already spread to the central nervous system (CNS). Using this approach we found 13 additional sheep with PrP(Sc) depositions in the gut-associated lymph nodes (GALT) as well as in the enteric nervous system. Moreover, in most of these cases PrP(Sc) was also deposited in the spleen and in the retropharyngeal and superficial cervical lymph nodes. Taken together, these results show a 30.3% infection prevalence in this scrapie-affected flock. Almost 7.4% of the infected animals harboured PrP(Sc) exclusively in the peripheral lymphatic and nervous tissue and were therefore missed by the currently used testing strategy.  相似文献   

18.
An immunohistochemical (IHC) method was used to test brain tissues from 17 elk in a captive herd in which chronic wasting disease (CWD) had previously occurred. The IHC technique detects the protease-resistant prion protein (PrP-res), which is considered a disease-specific marker for transmissible spongiform encephalopathies (TSE), regardless of the species affected. Of the 17 elk tested, 10 were positive by IHC. Only 2 of these 10 animals had shown clinical signs and histologic lesions of CWD, and an additional animal had histologic lesions only. The most consistently IHC-positive tissue was medulla oblongata, especially the obex. These results show that the PrP-res IHC test on brain tissue, specifically medulla oblongata at the obex, should be considered an essential component of any surveillance study intended to determine the incidence of CWD in captive or free-ranging cervids.  相似文献   

19.
Nor98 is an atypical scrapie strain characterized by a molecular pattern and brain distribution of the pathological prion protein (PrPSc) different from classical scrapie. In Italy, 69 atypical cases have been identified so far and all were characterized as Nor98 strain. In this paper we report an unusual case in a sheep which showed immunohistochemical and molecular features of PrPSc different from the other atypical cases. The sheep was from an outbreak where the index and the other four cases were affected by classical scrapie. Histopathological, immunohistochemical and Western blot analyses on the brain of the unusual case revealed the simultaneous presence of pathological features characteristic of Nor98 and classical scrapie. Interestingly, the prevalent disease phenotype in the brainstem was classical scrapie-like, while in the cerebral cortex and cerebellum the Nor98 phenotype was dominant. The sub-mandibular lymph node was positive and showed a PrPSc molecular pattern referable to classical scrapie. The PrP genotype was AL141RQ/AF141RQ. Taken together, the occurrence of classical scrapie in the outbreak, the PrP genotype, the involvement of different cellular targets in the brain and the pathological and molecular PrPSc features observed suggest that this unusual case may result from the co-existence of Nor98 and classical scrapie.  相似文献   

20.
This study determined whether the immunoassay for cerebrospinal fluid 14-3-3 protein concentration was sensitive and specific in the diagnosis of naturally occurring clinical scrapie in sheep. Cerebrospinal fluid was collected from 9 sheep with the confirmed diagnosis of scrapie. Additionally, cerebrospinal fluid was collected from 13 clinically normal sheep, which originated from a closely monitored flock with no history of scrapie. Sensitivity and specificity were calculated using standard epidemiological methods. Cerebrospinal fluid immunoassay results did not differ significantly between positive and negative sheep. Test sensitivity varied from 0.55 to 0.66, depending on the choice of test endpoint. Test specificity varied from 0.30 to 0.77, depending on the choice of test endpoint. The 14-3-3 cerebrospinal fluid immunoassay appears to have no value in the diagnosis of clinical scrapie in sheep.  相似文献   

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