Determination of sheep prion gene polymorphisms from paraffin-embedded tissue.

Amino acid polymorphisms of the prion protein (PrP) greatly influence the susceptibility of sheep to scrapie. Selective breeding to increase the prevalence of PrP gene alleles associated with scrapie resistance is a flock management practice that is important for scrapie control programs. Determination of sheep PrP alleles typically has required extraction of DNA from host tissues that are freshly derived or stored frozen. We describe application of a DNA extraction procedure for formalin-fixed, paraffin-embedded tissues (PET) for the purpose of PCR amplification and nucleotide sequencing of relevant codons (136-171) of the sheep PrP gene. Tissues derived from 96 sheep were studied. The DNA sequence identity was confirmed in 87 of 94 matched samples of PET and frozen tissue specimens. DNA from brainstem PET of 2 sheep, from which fresh tissue was not available, was amplified and sequenced after formalin fixation for 7-70 days. This method will allow retrospective analysis of PrP genetics of sheep subsequent to postmortem diagnosis of scrapie when nonfixed tissue is unavailable for DNA extraction; however, it is not recommended that submission of fixed tissue supplant collection of fresh tissues for the purpose of determining PrP gene polymorphisms.     

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.     

Mapping PrPSc propagation in experimental and natural scrapie in sheep with different PrP genotypes

Twenty-one orally inoculated and seven naturally infected sheep with scrapie were examined for PrP(Sc) in peripheral tissues and in the central nervous system (CNS), using immunohistochemistry. In the inoculated group, VRQ (valine at codon 136, arginine at codon 154 and glutamine at codon 171)/VRQ sheep generally had a greater accumulation of the pathologic form of prion protein (PrP(Sc)) in peripheral tissues, as compared with VRQ/ARQ (alanine at codon 136, arginine at codon 154, and glutamine at codon 171) animals at corresponding time points after inoculation. PrP(Sc) was not detected in the ileal Peyer's patch, the spleen, the superficial cervical lymph node, and peripheral nervous tissues of several inoculated VRQ/ARQ animals. All inoculated VRQ/VRQ sheep, but only one of eight inoculated VRQ/ARQ animals, were PrP(Sc)-positive in the CNS. Thus, the propagation of PrP(Sc) seemed slower and more limited in VRQ/ARQ animals. Tissue and cellular localization of PrP(Sc) suggested that PrP(Sc) was disseminated through three different routes. PrP(Sc)-positive cells in lymph node sinuses and in lymphatics indicated spreading by lymph. The sequential appearance of PrP(Sc) in the peripheral nervous system and the CNS, with satellite cells as early targets, suggested the periaxonal transportation of PrP(Sc) through supportive cells. Focal areas of vascular amyloid-like PrP(Sc) in the brain of five sheep, suggested the hematogenous dissemination of PrP(Sc). There was a poor correlation between the amount of PrP(Sc) in the CNS and clinical signs. One subclinically affected sheep showed widespread PrP(Sc) accumulation in the CNS, whereas three sheep had early clinical signs without detectable PrP(Sc) in the CNS. A VV(136) (homozygous for valine at codon 136) sheep inoculated with ARQ/ARR (alanine at codon 136, arginine at codon 154, and arginine at codon 171) tissue succumbed to disease, demonstrating successful heterologous transmission. Less susceptible sheep receiving VRQ/VRQ or ARQ/ARR material were PrP(Sc)-negative by immunohistochemistry, enzyme-linked immunosorbent assay, and western blot.     

Susceptibility of transgenic mice expressing chimeric sheep,bovine and human PrP genes to sheep scrapie

The use of Transgenic (Tg) mice expressing chimeric sheep/mouse (Sh/Mo) prion protein (PrP) and chimeric bovine/mouse (Bo/Mo) PrP genes was evaluated as a sheep scrapie model. We also investigated the potential for the transmission of sheep scrapie to a human/mouse (Hu/Mo) PrP Tg mouse line. The Sh/Mo PrP and Bo/Mo PrP Tg Prnp(+/+) or Prnp(0/0) mouse lines were inoculated intracerebrally with brain homogenates from three sheep with natural scrapie (KU, Y5 or S2). Incubation periods were slightly shorter in Sh/Mo PrP Tg Prnp(+/+), than in non-Tg mice inoculated with KU brain homogenate. In contrast, the incubation period was significantly prolonged (p<0.05) in Bo/Mo PrP Tg Prnp(+/+) mice inoculated with KU brain homogenate. The incubation period was significantly longer in all Tg Prnp(+/+) and Prnp(0/0), than in non-Tg mice (p<0.01) inoculated withY5 brain homogenate. None of the Tg Prnp(0/0) mice inoculated with S2 brain homogenate developed clinical signs and PrP(Sc) was undetectable in their brains. These results suggested that expression of the Sh/Mo PrP or Bo/Mo PrP transgenes does not confer susceptibility to sheep prions upon mice, and thus none of the Tg mouse lines could be a suitable model of sheep scrapie. Hu/Mo PrP Tg Prnp(0/0) mice inoculated with natural and experimental scrapie or mouse prions did not develop clinical signs of scrapie and PrP(Sc) was undetectable. These results suggested that neither sheep nor mouse strains of scrapie are highly transmissible to humans.     

In situ detection of cellular and abnormal isoforms of prion protein in brains of cattle with bovine spongiform encephalopathy and sheep with scrapie by use of a histoblot technique.

To detect prion protein, brains from 5 cattle naturally affected with bovine spongiform encephalopathy (BSE) and 3 sheep naturally affected with scrapie were examined and compared with brains of normal cattle and sheep using a histoblot technique. The technique enabled the in situ distinctive detection of the cellular (PrP(C)) and abnormal (PrP(Sc)) isoforms of the prion protein. In BSE- or scrapie-affected brains, the Prp(C) signal decreased, especially in those areas where the PrP(Sc) signal was detected.     

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.     

Unique amino acid polymorphisms of PrP genes in Mongolian sheep breeds

To characterize amino acid polymorphisms of sheep prion protein (PrP) gene, DNA from 740 sheep of nine breeds raised in Mongolia was isolated and analyzed. A total of 16 genotypes and seven allelic variants of the PrP gene at codons 112, 136, 154, and 171 were found. The MARQ/MARQ genotype associated with susceptibility to scrapie was found in 82.6% of the sheep while the MARR/MARR genotype associated with resistance to scrapie was found in 1.8% of the sheep. The polymorphisms of valine and serine at codon 127, and leucine and arginine at codon 189 were detected in eight Mongolian sheep breeds, suggesting that these polymorphisms are a common feature among Mongolian sheep breeds.     

Atypical scrapie in a Swiss goat and implications for transmissible spongiform encephalopathy surveillance.

Different types of transmissible spongiform encephalopathies (TSEs) affect sheep and goats. In addition to the classical form of scrapie, both species are susceptible to experimental infections with the bovine spongiform encephalopathy (BSE) agent, and in recent years atypical scrapie cases have been reported in sheep from different European countries. Atypical scrapie in sheep is characterized by distinct histopathologic lesions and molecular characteristics of the abnormal scrapie prion protein (PrP(sc)). Characteristics of atypical scrapie have not yet been described in detail in goats. A goat presenting features of atypical scrapie was identified in Switzerland. Although there was no difference between the molecular characteristics of PrP(sc) in this animal and those of atypical scrapie in sheep, differences in the distribution of histopathologic lesions and PrP(sc) deposition were observed. In particular the cerebellar cortex, a major site of PrP(sc) deposition in atypical scrapie in sheep, was found to be virtually unaffected in this goat. In contrast, severe lesions and PrP(sc) deposition were detected in more rostral brain structures, such as thalamus and midbrain. Two TSE screening tests and PrP(sc) immunohistochemistry were either negative or barely positive when applied to cerebellum and obex tissues, the target samples for TSE surveillance in sheep and goats. These findings suggest that such cases may have been missed in the past and could be overlooked in the future if sampling and testing procedures are not adapted. The epidemiological and veterinary public health implications of these atypical cases, however, are not yet known.     

Evaluation of the effects of controlled autolysis on the immunodetection of PrP(Sc) by immunoblotting and immunohistochemistry from natural cases of scrapie and BSE

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.     

Pre-clinical and clinical diagnosis of scrapie by detection of PrP protein in tissues of sheep.

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.     

Natural scrapie in British sheep: breeds, ages and PrP gene polymorphisms.

One hundred and sixty-seven sheep of 32 breeds and crossbreeds affected by natural scrapie throughout Britain were tested for the presence of restriction fragment length polymorphisms of the PrP gene observed when their DNA was digested with EcoRI or HindIII. These polymorphisms have already been associated with different susceptibilities to experimental scrapie (controlled by alleles of the Sip gene) in a flock of Cheviot sheep. In two studies 86 to 92 per cent of the sheep were found to carry the PrP gene EcoRI fragment e1 which is associated with high susceptibility (or the sA allele of Sip) to experimental scrapie. The PrP gene HindIII genotypes of the natural scrapie sheep were not apparently associated with differences in susceptibility to scrapie. There was no link between the polymorphisms and the age or breed of the affected sheep.     

Clinical and pathological features of experimental scrapie in Irish Blackface Mountain sheep

There have been no reports of natural scrapie in Irish Blackface Mountain (BM) sheep which account for approximately 16% of the Irish national sheep flock. The aim of this study was to determine if Irish BM sheep had unusual clinical and/or pathological features of scrapie which would account for failure to diagnose the disease in this breed. BM (n=7), Texel (n=3) and Suffolk sheep (n=1) of scrapie-susceptible PrP genotypes (ARQ/ARQ and VRQ/ARQ) were orally challenged with scrapie-infected brain inoculum. The incubation period, clinical signs, pathology and distribution of disease specific prion protein (PrP(d)) in scrapie-affected BM sheep were similar to scrapie in the Texel and Suffolk sheep. It was concluded that there was no evidence to suggest that scrapie in BM sheep differs clinicopathologically from scrapie in other breeds of sheep.     

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.     

Postmortem diagnosis of preclinical and clinical scrapie in sheep by the detection of disease-associated PrP in their rectal mucosa

Samples of tissue from the central nervous system (cns), the lymphoreticular system (lrs) and the rectal mucosa of a large number of scrapie-exposed sheep, with and without signs of clinical disease, were examined immunohistochemically for evidence of disease-associated prion protein (PrP(d)). The rectal mucosa has received almost no attention so far in scrapie diagnosis, despite its abundant rectoanal mucosa-associated lymphoid tissue, and its accessibility. The scrapie-confirmed cases included 244 with clinical disease, of which 237 (97.1 per cent) were positive in the rectal mucosa, and 121 apparently healthy sheep, of which 104 (86 per cent) were positive in the rectal mucosa. PrP(d) was detected in 86.4 to 91.5 per cent of the other lrs tissues of the healthy sheep examined and in 77.7 per cent of their cns tissues. The stage of infection, therefore, affected the probability of a positive result in the rectal mucosa, whereas the breed, PrP genotype, age and sex had little or no independent effect. Accumulations of PrP(d) were observed in the rectal mucosa and other lrs tissues of vrq/arr sheep with preclinical and clinical scrapie, albeit with a lower frequency and magnitude than in sheep of other PrP genotypes. Western immunoblotting analyses of samples of rectal mucosa gave the characteristic PrP glycoprofile, with a sensitivity similar to that of immunohistochemistry.     

Preliminary observations on the experimental transmission of scrapie to elk (Cervus elaphus nelsoni) by intracerebral inoculation

To determine the transmissibility of scrapie to Rocky Mountain elk (Cervus elaphus nelsoni), six elk calves were inoculated intracerebrally with brain suspension from sheep naturally affected with scrapie. One elk developed a brain abscess and was euthanatized at 7 weeks postinoculation (PI), and two others died at 6 and 15 months PI because of physical injuries. At 25 and 35 months PI, two other elk died after brief terminal neurologic episodes. Necropsy of these revealed moderate weight loss but no other gross lesions. Microscopically, characteristic lesions of spongiform encephalopathy were seen throughout the brains and the spinal cords, and in both cases these tissues were positive for PrP(res) by immunohistochemistry. Brains of both animals were positive for PrP(res) by western blot and for scrapie-associated fibrils (SAFs) by negative stain electron microscopy. PrP(res) and SAFs were not detected in the three elk that died or were euthanatized because of coincidental causes. Over 3.5 years after initiation of this experiment, the one remaining inoculated elk and two uninoculated (control) elk are alive and apparently healthy. These preliminary findings demonstrate that 1) sheep scrapie agent can be transmitted to elk by intracerebral inoculation; 2) the infection can result in severe, widely distributed spongiform change and accumulations of PrP(res) in the central nervous system (CNS); and 3) based on the examination of a limited number of CNS sections from two cases, this condition cannot be distinguished from chronic wasting disease with currently available diagnostic techniques.     

Degradation of scrapie associated prion protein (PrPSc) by the gastrointestinal microbiota of cattle

A food-borne origin of the transmission of bovine spongiform encephalopathy (BSE) to cattle is commonly assumed. However, the fate of infectious prion protein during polygastric digestion remains unclear. It is unknown at present, whether infectious prion proteins, considered to be very stable, are degraded or inactivated by microbial processes in the gastrointestinal tract of cattle. In this study, rumen and colon contents from healthy cattle, taken immediately after slaughter, were used to assess the ability of these microbial consortia to degrade PrP(Sc). Therefore, the consortia were incubated with brain homogenates of scrapie (strain 263K) infected hamsters under physiological anaerobic conditions at 37 degrees C. Within 20 h, PrP(Sc) was digested both with ruminal and colonic microbiota up to immunochemically undetectable levels. Especially polymyxin resistant (mainly gram-positive) bacteria expressed PrP(Sc) degrading activity. These data demonstrate the ability of bovine gastrointestinal microbiota to degrade PrP(Sc) during digestion.     

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。     

A survey of more than 11 years of neurologic diseases of ruminants with special reference to transmissible spongiform encephalopathies (TSEs) in Greece

The first cases of scrapie were detected in Greece in a flock of sheep in October 1986. All the animals of the affected flock and all sheep in two flocks that were in contact were killed and buried. A systematic investigation of all available cases with signs indicating a neurological disease started in sheep and goats in late 1986, as well as in cattle in 1989. The investigation was based on clinical examination, necropsy or macroscopical examination of the brain and viscera, and histological examination of the brain in all animals except those with coenurosis. Histological examinations of specimens from the spinal cord and other tissues, and if considered necessary bacteriological, toxicological and serological examinations were also carried out. In October 1997, scrapie was diagnosed in sheep of a second flock (a mixed flock of sheep and goats), grazing in a pasture close to the place where scrapie was initially detected. All animals of the second flock were also killed and buried. Diagnosis in the first flock was based on clinical signs and histological lesions, and in the second immunoblotting was also used. Distinctive lesions of scrapie were found in the brain and/or the spinal cord of eight sheep with clinical signs from the two flocks. The lesions were revealed in the brain stem and/or in the cervical spinal cord, and tended to be symmetrical. In one sheep, severe lesions in the cortex of cerebral hemispheres and of the cerebellum were also found. In the brain of two sheep from the second flock the pathological isoform of PrP protein was detected. Despite the eradication scheme applied, scrapie in sheep reappeared after 11 years in a place close to where it occurred initially. This may indicate that the effectiveness of the eradication scheme implemented was not adequate and additional approaches may be needed.     

Rapid testing leads to the underestimation of the scrapie prevalence in an affected sheep and goat flock

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.