Co-existence of classical scrapie and Nor98 in a sheep from an Italian outbreak

Nor98 is an atypical scrapie strain characterized by a molecular pattern and brain distribution of the pathological prion protein (PrP^Sc) 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 PrP^Sc 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 PrP^Sc 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 PrP^Sc features observed suggest that this unusual case may result from the co-existence of Nor98 and classical scrapie.     

Second passage of chronic wasting disease of mule deer to sheep by intracranial inoculation compared to classical scrapie

The origin of chronic wasting disease (CWD) in cervids is unclear. One hypothesis suggests that CWD originated from scrapie in sheep. We compared the disease phenotype of sheep-adapted CWD to classical scrapie in sheep. We inoculated sheep intracranially with brain homogenate from first-passage mule deer CWD in sheep (sCWD^md). The attack rate in second-passage sheep was 100% (12 of 12). Sheep had prominent lymphoid accumulations of PrP^Sc reminiscent of classical scrapie. The pattern and distribution of PrP^Sc in the brains of sheep with CWD^md was similar to scrapie strain 13-7 but different from scrapie strain x124. The western blot glycoprofiles of sCWD^md were indistinguishable from scrapie strain 13-7; however, independent of sheep genotype, glycoprofiles of sCWD^md were different than x124. When sheep genotypes were evaluated individually, there was considerable overlap in the glycoprofiles that precluded significant discrimination between sheep CWD and scrapie strains. Our data suggest that the phenotype of CWD in sheep is indistinguishable from some strains of scrapie in sheep. Given our results, current detection techniques would be unlikely to distinguish CWD in sheep from scrapie in sheep if cross-species transmission occurred naturally. It is unknown if sheep are naturally vulnerable to CWD; however, the susceptibility of sheep after intracranial inoculation and lymphoid accumulation indicates that the species barrier is not absolute.     

Pathological characterization of TgElk mice injected with brain homogenate from elk with chronic wasting disease

Chronic wasting disease (CWD) is classified as a transmissible spongiform encephalopathy or prion disease that affects cervids. CWD has been reported in 15 US states, two Canadian provinces, and in imported elk on several farms in Korea. This study was conducted to examine the molecular biological and pathogenic characteristics of a CWD-associated prion isolated in Korea. The epidemiological origin of this pathogen was also determined. Homozygous TgElk mice were infected with a CWD-affected elk brain pool prepared from the brain of an imported Canadian elk. We measured the incubation time of the pathogen, neuropathological changes by immunohistochemical staining, the pattern(s) of scrapie prion protein (PrP^Sc) deposition, and PrP^Sc protein profiles by Western blotting. We found that TgElk mice infected with brain homogenate from the elk suffering from CWD showed incubation times, vacuolar degeneration, and PrP^Sc accumulation similar to those previously reported in the literature. Our results suggest that homozygous TgElk mice efficiently transmit CWD with short incubation times and that this animal can serve a valuable research model and reliable in vivo diagnostic tool.     

Classical natural ovine scrapie prions detected in practical volumes of blood by lamb and transgenic mouse bioassays

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.     

Incubation of ovine scrapie with environmental matrix results in biological and biochemical changes of PrPSc over time

Ovine scrapie can be transmitted via environmental reservoirs. A pool of ovine scrapie isolates were incubated on soil for one day or thirteen months and eluted prion was used to challenge tg338 mice transgenic for ovine PrP. After one-day incubation on soil, two PrP^Sc phenotypes were present: G₃₃₈ or Apl₃₃₈ii. Thirteen months later some divergent PrP^Sc phenotypes were seen: a mixture of Apl₃₃₈ii with either G₃₃₈ or P_338, and a completely novel PrP^Sc deposition, designated Cag₃₃₈. The data show that prolonged ageing of scrapie prions within an environmental matrix may result in changes in the dominant PrP^Sc biological/biochemical properties.     

Failure to detect abnormal prion protein and scrapie-associated fibrils 6 wk after intracerebral inoculation of genetically susceptible sheep with scrapie agent

Detection of the scrapie-associated protease-resistant prion protein (PrP^res) in sheep brains in the early phase after intracerebral inoculation of the scrapie agent has not been documented. Fourteen 4-mo-old, genetically susceptible lambs (QQ homozygous at codon 171 of the PrP gene) were obtained for this study. Twelve lambs were inoculated intracerebrally with a brain suspension from sheep naturally affected with scrapie, and 2 served as uninoculated controls. Two inoculated animals were euthanized at each of 6 times postinoculation (1 h to 6 wk), and their brains were collected for histopathological study, for detection of PrP^res by the Western blot technique and an immunohistochemical (IHC) method, and for the detection of scrapie-associated fibrils (SAF) by negatively stained electron microscopy (EM). Microscopic lesions associated with introduction of the inoculum were seen in the brains of inoculated animals at all 6 times. However, both the Western blot and IHC techniques did not detect PrP^res after the initial 3 d postinoculation, nor did EM detect SAF in any of the samples. From these findings, it is presumed that until host amplification has occurred, the concentration of PrP^res in inoculum is insufficient for detection by currently available techniques.     

Experimental infection of meadow voles (Microtus pennsylvanicus) with sheep scrapie

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 (PrP^res) 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 (PrP^Sc) deposition were generally similar in all scrapie-affected voles, except in the hippocampus, where PrP^Sc 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.     

Observations on thermostable subpopulations of the unconventional agents that cause transmissible degenerative encephalopathies

When scrapie agent is exposed to partially inactivating autoclave cycles, the fraction of infectivity that survives remains thermostable during relatively long periods of autoclaving. This resistant subpopulation can also be differentiated from the main population by its prolonged incubation periods in assay animals, compared with control material. Stabilisation of this subpopulation may occur through the smearing and drying of infected tissue that can occur prior to autoclaving, in which the disease-specific form of PrP protein (PrP^Sc) could become rapidly heat-fixed. This may paradoxically be what protects this fraction of PrP^Sc from further inactivation during autoclaving. Data are presented showing that the thermostability acquired by the resistant subpopulation is a stable characteristic; autoclaving for a second time results in very little further loss of infectivity. These observations suggest that inactivation procedures that do not involve rapid and effective fixation of PrP^Sc may be better candidates for dealing effectively with scrapie-like agents.     

Detection of PrPSc in lung and mammary gland is favored by the presence of Visna/maedi virus lesions in naturally coinfected sheep

There are few reports on the pathogenesis of scrapie (Sc) and Visna/maedi virus (VMV) coinfections. The aim of this work was to study in vivo as well as post mortem both diseases in 91 sheep. Diagnosis of Sc and VMV infections allowed the distribution of animals into five groups according to the presence (+) or absence (−) of infection by Sc and VMV: Sc−/VMV−, Sc−/VMV+, Sc+/VMV− and Sc+/VMV+. The latter was divided into two subgroups, with and without VMV-induced lymphoid follicle hyperplasia (LFH), respectively. In both the lung and mammary gland, PrP^Sc deposits were found in the germinal center of hyperplasic lymphoid follicles in the subgroup of Sc+/VMV+ having VMV-induced LFH. This detection was always associated with (and likely preceded by) PrP^Sc observation in the corresponding lymph nodes. No PrP^Sc was found in other VMV-associated lesions. Animals suffering from scrapie had a statistically significantly lower mean age than the scrapie free animals at the time of death, with no apparent VMV influence. ARQ/ARQ genotype was the most abundant among the 91 ewes and the most frequent in scrapie-affected sheep. VMV infection does not seem to influence the scrapie risk group distribution among animals from the five groups established in this work. Altogether, these data indicate that certain VMV-induced lesions can favor PrP^Sc deposits in Sc non-target organs such as the lung and the mammary gland, making this coinfection an interesting field that warrants further research for a better comprehension of the pathogenesis of both diseases.     

Cellular prion proteins in humans and cattle but not sheep are characterized by a low-solubility phenotype

A feature of transmissible spongiform encephalopathies is the accumulation of infectious prion proteins (PrP^Sc), which are formed by the conversion of physiological prion proteins (PrP^C). As PrP^C, which is modified posttranslationally with various types of glycoproteins, serves as the substrates for PrP^Sc conversion, various PrP^C subtypes may play a role in the formation of PrP^Sc and species-specific transmission; the cattle disease BSE is transmissible naturally to humans, but the sheep disease scrapie is not. To reveal new mechanisms modulating prion conversion, we analyzed the PrP^C profiles by determining the differential PrP^C protein solubilities in the anionic and nonionic detergents N-lauroylsarcosine, N-octyl-β-d-glucopyranoside, CHAPS and deoxycholic acid. We compared the resulting solubility profiles of human PrP^C with the solubility profiles of PrP^C from sheep and cattle. The PrP^C subtypes were differentially soluble. However, non-glycosylated PrP^C from cattle and human was found explicitly in the insoluble fraction, while non-glycosylated ovine PrP^C was detected in the soluble fraction. These findings indicate the existence of low-solubility PrP^C phenotypes in cattle and humans.     

Circulation of prions within dust on a scrapie affected farm

Prion diseases are fatal neurological disorders that affect humans and animals. Scrapie of sheep/goats and Chronic Wasting Disease (CWD) of deer/elk are contagious prion diseases where environmental reservoirs have a direct link to the transmission of disease. Using protein misfolding cyclic amplification we demonstrate that scrapie PrP^Sc can be detected within circulating dusts that are present on a farm that is naturally contaminated with sheep scrapie. The presence of infectious scrapie within airborne dusts may represent a possible route of infection and illustrates the difficulties that may be associated with the effective decontamination of such scrapie affected premises.     

Hepatitis in dogs. An indexed bibliography of journal articles (1994–2004)

Transmissible spongiform encephalopathies (TSEs) or prion diseases are unique disorders that are not caused by infectious micro-organisms (bacteria or fungi), viruses or parasites, but rather seem to be the result of an infectious protein. TSEs are comprised of fatal neurodegenerative disorders affecting both human and animals. Prion diseases cause sponge-like degeneration of neuronal tissue and include (among others) Creutzfeldt–Jacob disease in humans, bovine spongiform encephalopathy (BSE) in cattle and scrapie in sheep. TSEs are characterized by the formation and accumulation of transmissible (infectious) disease-associated protease-resistant prion protein (PrP^Sc), mainly in tissues of the central nervous system. The exact molecular processes behind the conversion of PrP^C into PrP^Sc are not clearly understood. Correlations between prion protein polymorphisms and disease have been found, however in what way these polymorphisms influence the conversion processes remains an enigma; is stabilization or destabilization of the prion protein the basis for a higher conversion propensity? Apart from the disease-associated polymorphisms of the prion protein, the molecular processes underlying conversion are not understood. There are some notions as to which regions of the prion protein are involved in refolding of PrP^C into PrP^Sc and where the most drastic structural changes take place. Direct interactions between PrP^C molecules and/or PrP^Sc are likely at the basis of conversion, however which specific amino acid domains are involved and to what extent these domains contribute to conversion resistance/sensitivity of the prion protein or the species barrier is still unknown.     

Susceptibility to scrapie and disease phenotype in sheep: cross-PRNP genotype experimental transmissions with natural sources

It has long been established that the sheep Prnp genotype influences the susceptibility to scrapie, and some studies suggest that it can also determine several aspects of the disease phenotype. Other studies, however, indicate that the source of infection may also play a role in such phenotype. To address this question an experiment was set up in which either of two different natural scrapie sources, AAS from AA₁₃₆ Suffolk and VVC from VV₁₃₆ Cheviot sheep, were inoculated into AA₁₃₆, VA₁₃₆ and VV₁₃₆ sheep recipients (n = 52). The immunohistochemical (IHC) profile of disease-associated PrP (PrP^d) accumulation in the brain of recipient sheep was highly consistent upon codon 136 homologous and semi-homologous transmission, but could be either similar to or different from those of the inoculum donors. In contrast, the IHC profiles were highly variable upon heterologous transmission (VVC to AA₁₃₆ and AAS to VV₁₃₆). Furthermore, sheep of the same Prnp genotype could exhibit different survival times and PrP^d profiles depending on the source of infection, and a correlation was observed between IHC and Western blot profiles. It was found that additional polymorphisms at codons 112 or 141 of AA₁₃₆ recipients resulted in a delayed appearance of clinical disease or even in protection from infection. The results of this study strongly suggest that the scrapie phenotype in sheep results from a complex interaction between source, donor and recipient factors, and that the Prnp genotype of the recipient sheep does not explain the variability observed upon codon 136 heterologous transmissions, arguing for other genetic factors to be involved.     

Sensitivity and specificity of a commercial BSE kit for the detection of ovine scrapie

To examine the sensitivity of a commercially available bovine spongiform encephalopathy (BSE) kit (NippIBL) for the detection of ovine scrapie, 50 scrapie‐positive ovine samples from the UK, and 54 scrapie‐negative ovine samples from Japan were obtain and tested using this kit. The sensitivity and specificity of NippIBL for ovine samples were 96% and 100%, respectively. The detection limit of the abnormal isoform of prion protein (PrP^Sc) of NippIBL was examined using diluted scrapie‐positive samples. The sensitivity of NippIBL to ovine scrapie was 3–10 times superior to that of another commercial BSE diagnosis kit. Thus, the NippIBL kit proved more effective for the detection of ovine scrapie.     

Complementary studies detecting classical bovine spongiform encephalopathy infectivity in jejunum,ileum and ileocaecal junction in incubating cattle

Recently we have described the distribution of bovine spongiform encephalopathy (BSE) infectivity and/or PrP^Sc in Peyer’s patches (PP) of the small intestine of orally BSE infected cattle. In this follow-up study additional jejunal and ileal PP’s and ileocaecal-junction tissue samples from 1, 4, and 24 months post infection (mpi) were examined by mouse (Tgbov XV) bioassay. Infectivity was demonstrated in ileal PP’s 4 mpi and the distribution/extent of infectivity at 24 mpi was comparable to those seen at earlier time points, revealing no indication for a decline/clearance. These data are relevant for the definition of Specified Risk Materials in the context of the TSE legislation worldwide.     

Acquired transmissibility of sheep-passaged L-type bovine spongiform encephalopathy prion to wild-type mice

L-type bovine spongiform encephalopathy (L-BSE) is an atypical form of BSE that is transmissible to cattle and several lines of prion protein (PrP) transgenic mice, but not to wild-type mice. In this study, we examined the transmissibility of sheep-passaged L-BSE prions to wild-type mice. Disease-associated prion protein (PrP^Sc) was detected in the brain and/or lymphoid tissues during the lifespan of mice that were asymptomatic subclinical carriers, indicating that wild-type mice were susceptible to sheep-passaged L-BSE. The morphological characteristics of the PrP^Sc of sheep-passaged L-BSE included florid plaques that were distributed mainly in the cerebral cortex and hippocampus of subsequent passaged mice. The PrP^Sc glycoform profiles of wild-type mice infected with sheep-passaged L-BSE were similar to those of the original isolate. The data indicate that sheep-passaged L-BSE has an altered host range and acquired transmissibility to wild-type mice.     

The Potential of Mesenchymal Stem Cell in Prion Research

Scrapie and bovine spongiform encephalopathy are fatal neurodegenerative diseases caused by the accumulation of a misfolded protein (PrP^res), the pathological form of the cellular prion protein (PrP^C). For the last decades, prion research has greatly progressed, but many questions need to be solved about prion replication mechanisms, cell toxicity, differences in genetic susceptibility, species barrier or the nature of prion strains. These studies can be developed in murine models of transmissible spongiform encephalopathies, although development of cell models for prion replication and sample titration could reduce economic and timing costs and also serve for basic research and treatment testing. Some murine cell lines can replicate scrapie strains previously adapted in mice and very few show the toxic effects of prion accumulation. Brain cell primary cultures can be more accurate models but are difficult to develop in naturally susceptible species like humans or domestic ruminants. Stem cells can be differentiated into neuron‐like cells and be infected by prions. However, the use of embryo stem cells causes ethical problems in humans. Mesenchymal stem cells (MSCs) can be isolated from many adult tissues, including bone marrow, adipose tissue or even peripheral blood. These cells differentiate into neuronal cells, express PrP^C and can be infected by prions in vitro. In addition, in the last years, these cells are being used to develop therapies for many diseases, including neurodegenerative diseases. We review here the use of cell models in prion research with a special interest in the potential use of MSCs.     

Molecular profiling and comparison of field transmissible spongiform encephalopathy cases diagnosed in Catalunya

Molecular profiling of the proteinase K resistant prion protein (PrP(res)) is a technique that has been applied to the characterisation of transmissible spongiform encephalopathy (TSE) strains. An interesting example of the application of this technique is the ability to differentiate, at the experimental level, between bovine spongiform encephalopathy (BSE) and scrapie infection in sheep, and to distinguish between classical and atypical BSE and scrapie cases. Twenty-six BSE cases and two scrapie cases from an active TSE surveillance program and diagnosed at the PRIOCAT, TSE Reference Laboratory (Centre de Recerca en Sanitat Animal, Universitat Autònoma de Barcelona, Catalunya, Spain) were examined by Western blotting. Molecular profiling was achieved by comparing the glycosylation profile, deglycosylated PrP molecular weight and 6H4/P4 monoclonal antibody binding ratio. The results obtained during the characterisation of these field cases indicated an absence of atypical BSE cases in Catalunya.