Alternating anti-prion regimens reduce combination drug resistance but do not further extend survival in scrapie-infected mice.

Prion diseases are fatal and infectious neurodegenerative diseases in humans and other mammals caused by templated misfolding of the endogenous prion protein (PrP). Although there is currently no vaccine or therapy against prion disease, several classes of small-molecule compounds have been shown to increase disease-free incubation time in prion-infected mice. An apparent obstacle to effective anti-prion therapy is the emergence of drug-resistant strains during static therapy with either single compounds or multi-drug combination regimens. Here, we treated scrapie-infected mice with dynamic regimens that alternate between different classes of anti-prion drugs. The results show that alternating regimens containing various combinations of Anle138b, IND24 and IND116135 reduce the incidence of combination drug resistance, but do not significantly increase long-term disease-free survival compared to monotherapy. Furthermore, the alternating regimens induced regional vacuolation profiles resembling those generated by a single component of the alternating regimen, suggesting the emergence of strain dominance.

Autoclave treatment of the classical scrapie agent US No. 13-7 and experimental inoculation to susceptible VRQ/ARQ sheep via the oral route results in decreased transmission efficiency.

Scrapie, a prion disease of sheep, is highly resistant to conventional deactivation. Numerous methods to deactivate scrapie have been tested in laboratory animal models, and adequate autoclave treatment can reduce or remove the infectivity of some classical scrapie strains depending on the heating parameters used. In this study, we autoclaved brain homogenate from a sheep with US scrapie strain 13-7 for 30 minutes at 121°C. Genetically susceptible VRQ/ARQ sheep were orally inoculated with 3 grams of the autoclaved brain homogenate. For comparison, a second group of sheep was inoculated with a non-autoclaved brain homogenate. Rectal biopsies were used to assess antemortem scrapie disease progression throughout the study. Five out of ten (5/10) sheep that received autoclaved inoculum ultimately developed scrapie after an experimental endpoint of 72 months. These sheep had a mean incubation period of 26.99 months. Two out of five (2/5) positive sheep had detectable PrPSc in antemortem rectal biopsies, and two (2/5) other sheep had PrPSc in postmortem rectal tissue. A single sheep (1/5) was positive for scrapie in the CNS, small intestine, and retropharyngeal lymph node but had negative rectal tissue. All of the sheep (10/10) that received non-autoclaved inoculum developed scrapie with a mean incubation period of 20.2 months and had positive rectal biopsies at the earliest timepoint (14.7 months post-inoculation). These results demonstrate that sheep are orally susceptible to US derived classical scrapie strain 13-7 after autoclave treatment at 121°C for 30 minutes. Differences in incubation periods and time interval to first positive rectal biopsies indicate a partial reduction in infectivity titers for the autoclaved inoculum group.

Lymphocyte activation gene 3 (Lag3) expression is increased in prion infections but does not modify disease progression.

Prion diseases, Alzheimer's disease and Parkinson's disease (PD) are fatal degenerative disorders that share common neuropathological and biochemical features, including the aggregation of pathological protein conformers. Lymphocyte activation gene 3 (Lag3, also known as CD223) is a member of the immunoglobulin superfamily of receptors expressed on peripheral immune cells, microglia and neurons, which serves as a receptor for α-synuclein aggregates in PD. Here we examined the possible role of Lag3 in the pathogenesis of prion diseases. Through quantitative real-time PCR and RNA-sequencing, we found that the expression levels of Lag3 were relatively low in the adult mouse brains, yet its expression was increased after prion infection. However, we failed finding significant differences regarding the incubation time, PrPSc load, neurodegeneration, astrocyte and microglia reactions and inflammatory gene expression between the Lag3 knockout mice and wild-type littermate controls after prion infection. We conclude that loss of Lag3 has no significant influence on prion disease pathogenesis. Considering that Lag3 is an immune checkpoint receptor, our results suggest that immune checkpoint inhibition (an increasingly prevalent therapeutic modality against many types of cancer) might not exert positive or negative effects on the progression of prion diseases.

Phosphorylated human tau associates with mouse prion protein amyloid in scrapie-infected mice but does not increase progression of clinical disease.

Tauopathies are a family of neurodegenerative diseases in which fibrils of human hyperphosphorylated tau (P-tau) are believed to cause neuropathology. In Alzheimer disease, P-tau associates with A-beta amyloid and contributes to disease pathogenesis. In familial human prion diseases and variant CJD, P-tau often co-associates with prion protein amyloid, and might also accelerate disease progression. To test this latter possibility, here we compared progression of amyloid prion disease in vivo after scrapie infection of mice with and without expression of human tau. The mice used expressed both anchorless prion protein (PrP) and membrane-anchored PrP, that generate disease associated amyloid and non-amyloid PrP (PrPSc) after scrapie infection. Human P-tau induced by scrapie infection was only rarely associated with non-amyloid PrPSc, but abundant human P-tau was detected at extracellular, perivascular and axonal deposits associated with amyloid PrPSc. This pathology was quite similar to that seen in familial prion diseases. However, association of human and mouse P-tau with amyloid PrPSc did not diminish survival time following prion infection in these mice. By analogy, human P-tau may not affect prion disease progression in humans. Alternatively, these results might be due to other factors, including rapidity of disease, blocking effects by mouse tau, or low toxicity of human P-tau in this model.

Potential approaches for heterologous prion protein treatment of prion diseases.

Prion diseases, or transmissible spongiform encephalopathies (TSEs) are progressive, fatal neurodegenerative diseases with no effective treatment. The pathology of these diseases involves the conversion of a protease sensitive form of the cellular prion protein (PrP(C)) into a protease resistant infectious form (PrP(res)). The efficiency of this conversion is predicated upon a number of factors, most notably a strong homology between cellular PrP(C) and PrP(res). In our recently published study, we infected mice with the RML-Chandler strain of scrapie and treated them with heterologous hamster prion proteins. This treatment was seen to reduce clinical signs of prion disease, to delay the onset of clinical symptoms and to prolong survival. In this current article we discuss potential mechanisms of action of treatment with heterologous prion proteins. We also discuss potential extensions of these studies using a heterologous rabbit PrP-based treatment strategy or a peptide based strategy, and improvement of treatment delivery including a lentiviral-based system.

Diets with different lipid contents do not modify the neuronal membrane lipid raft profile in a scrapie murine model.

UNLABELLED: In Transmissible Spongiform Encephalopathies (TSEs), the localization of the prion protein in the neuronal membrane lipid rafts (LR) seems to play a role in sustaining the protein misfolding. Changes in membrane properties, due to altered lipid composition, affect their organization and interaction between lipids and protein therein, and consequently also membrane resident protein functionality; dietary polyunsaturated fatty acids (PUFAs), gangliosides and cholesterol seem to influence these processes. AIMS: In this work, the influence of administration of different feed, able to change the composition of lipid membrane, on the clinical progression of prion disease was studied. MAIN METHODS: The activity of three diets (hyperlipidic with 6% fats; hypolipidic with 0.1% fats; and purified with 4% fats) was tested in CD1 mouse model experimentally infected with RML scrapie strain. Presence and distribution of typical central nervous system (CNS) lesions and deposits of PrP(sc) were evaluated by histopathological analysis and immunohistochemistry. Analysis of lipids was performed in homogenate and insoluble brain fraction of the neuronal membrane rich in LR. KEY FINDINGS: Results show that a diet with a different lipid level has not a significant role in the development of the scrapie disease. All infected mice fed with different diets died in the same time span. Histology, immunohistochemistry, and neuropathological analyses of the infected brains did not show significant differences between animals subjected to different diets. SIGNIFICANCE: Independently of the diet, the infection induced a significant modification of the lipid composition in homogenates, and a less noticeable one in insoluble brain fraction.

Use of a 2-aminothiazole to Treat Chronic Wasting Disease in Transgenic Mice.

Treatment with the 2-aminothiazole IND24 extended the survival of mice infected with mouse-adapted scrapie but also resulted in the emergence of a drug-resistant prion strain. Here, we determined whether IND24 extended the survival of transgenic mice infected with prions that caused scrapie in sheep or prions that caused chronic wasting disease (CWD; hereafter "CWD prions") in deer, using 2 isolates for each disease. IND24 doubled the incubation times for mice infected with CWD prions but had no effect on the survival of those infected with scrapie prions. Biochemical, neuropathologic, and cell culture analyses were used to characterize prion strain properties following treatment, and results indicated that the CWD prions were not altered by IND24, regardless of survival extension. These results suggest that IND24 may be a viable candidate for treating CWD in infected captive cervid populations and raise questions about why some prion strains develop drug resistance whereas others do not.

Correlation between infectivity and disease associated prion protein in the nervous system and selected edible tissues of naturally affected scrapie sheep.

The transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of fatal neurodegenerative disorders characterised by the accumulation of a pathological form of a host protein known as prion protein (PrP). The validation of abnormal PrP detection techniques is fundamental to allow the use of high-throughput laboratory based tests, avoiding the limitations of bioassays. We used scrapie, a prototype TSE, to examine the relationship between infectivity and laboratory based diagnostic tools. The data may help to optimise strategies to prevent exposure of humans to small ruminant TSE material via the food chain. Abnormal PrP distribution/accumulation was assessed by immunohistochemistry (IHC), Western blot (WB) and ELISA in samples from four animals. In addition, infectivity was detected using a sensitive bank vole bioassay with selected samples from two of the four sheep and protein misfolding cyclic amplification using bank vole brain as substrate (vPMCA) was also carried out in selected samples from one animal. Lymph nodes, oculomotor muscles, sciatic nerve and kidney were positive by IHC, WB and ELISA, although at levels 100-1000 fold lower than the brain, and contained detectable infectivity by bioassay. Tissues not infectious by bioassay were also negative by all laboratory tests including PMCA. Although discrepancies were observed in tissues with very low levels of abnormal PrP, there was an overall good correlation between IHC, WB, ELISA and bioassay results. Most importantly, there was a good correlation between the detection of abnormal PrP in tissues using laboratory tests and the levels of infectivity even when the titre was low. These findings provide useful information for risk modellers and represent a first step toward the validation of laboratory tests used to quantify prion infectivity, which would greatly aid TSE risk assessment policies.

Successive passaging of the scrapie strains, ME7-ha and 139A-ha, generated by the interspecies transmission of mouse-adapted strains into hamsters markedly shortens the incubation times, but maintains their molecular and pathological properties.

As a type of zoonotic disease, prion diseases may be transmitted naturally and experimentally among species. In a previous study, we demonstrated that the mouse-adapted scrapie strains, ME7 (ME7-mo) and 139A (139A-mo), can overcome the species barrier and induce experimental scrapie when inoculated into Golden hamsters and generated 2 new hamster-adapted strains, ME7 (ME7-ha) and 139A (139A-ha). In the present study, in order to assess the infectivity and other molecular and neuropathological properties of the newly formed scrapie agents, ME7-ha and 139A-ha were further intracerebrally inoculated into hamsters. Compared with infection with 1st passage strains, the incubation times and clinical courses of infection with 2nd passage strains were markedly shorter, which were quite comparable with those of the mice infected with their parent mouse strains. The glycosylation patterns of brain PrP(Sc) in the animals infected with the 2nd passage of those 2 strains maintained similar features as those in the animals infected with the 1st passage of those strains, with predominantly diglycosylated PrP(Sc). Neuropathological assays revealed comparable spongiform degeneration and microglia proliferation in the brain tissues from the infected mice and hamsters, but markedly more plaque-like deposits of PrP(Sc) and more severe astrogliosis in the brains of the hamster. These data indicate that the strains, ME7-ha 1st and 139A-ha 1st generated by interspecies infection can passage in the new host hamster and stably maintain their molecular and neuropathological characteristics.

Role of the goat K222-PrP(C) polymorphic variant in prion infection resistance.

UNLABELLED: The prion protein-encoding gene (prnp) strongly influences the susceptibility of small ruminants to transmissible spongiform encephalopathies (TSEs). Hence, selective breeding programs have been implemented to increase sheep resistance to scrapie. For goats, epidemiological and experimental studies have provided some association between certain polymorphisms of the cellular prion protein (PrP(C)) and resistance to TSEs. Among them, the Q/K polymorphism at PrP(C) codon 222 (Q/K222) yielded the most promising results. In this work, we investigated the individual effects of the K222-PrP(C) variant on the resistance/susceptibility of goats to TSEs. For that purpose, we generated two transgenic mouse lines, expressing either the Q222 (wild type) or K222 variant of goat PrP(C). Both mouse lines were challenged intracerebrally with a panel of TSE isolates. Transgenic mice expressing the wild-type (Q222) allele were fully susceptible to infection with all tested isolates, whereas transgenic mice expressing similar levels of the K222 allele were resistant to all goat scrapie and cattle BSE isolates but not to goat BSE isolates. Finally, heterozygous K/Q222 mice displayed a reduced susceptibility to the tested panel of scrapie isolates. These results demonstrate a highly protective effect of the K222 variant against a broad panel of different prion isolates and further reinforce the argument supporting the use of this variant in breeding programs to control TSEs in goat herds. IMPORTANCE: The objective of this study was to determine the role of the K222 variant of the prion protein (PrP) in the susceptibility/resistance of goats to transmissible spongiform encephalopathies (TSEs). Results showed that transgenic mice expressing the goat K222-PrP polymorphic variant are resistant to scrapie and bovine spongiform encephalopathy (BSE) agents. This protective effect was also observed in heterozygous Q/K222 animals. Therefore, the single amino acid exchange from Q to K at codon 222 of the cellular prion protein provides resistance against TSEs. All the results presented here support the view that the K222 polymorphic variant is a good candidate for selective breeding programs to control and eradicate scrapie in goat herds.

Deletion of protease-activated receptor 2 prolongs survival of scrapie-inoculated mice.

Proteinase-activated receptor 2 (PAR2) has recently been identified to be a possible modulator of neurodegeneration. To investigate whether PAR2 plays a role in prion infection, we inoculated PAR2-deficient (PAR2(-/-)) and wild-type (WT) mice intracerebrally with the Rocky Mountain Laboratory strain of scrapie. PAR2(-/-) mice demonstrated a delayed onset of clinical symptoms, including weight loss, and demonstrated moderate but highly significant prolongation of survival over WT controls. Concomitantly, no apparent differences in brain pathology, infectivity or features of brain prion protein between deceased WT and PAR2(-/-) mice were found. Our study suggests that PAR2 deletion modulates dynamics of the disease without gross perturbation of its pathogenesis.

Mouse-adapted scrapie strains 139A and ME7 overcome species barrier to induce experimental scrapie in hamsters and changed their pathogenic features.

BACKGROUND: Transmissible spongiform encephalopathy (TSE) diseases are known to be zoonotic diseases that can infect different kinds of animals. The transmissibility of TSE, like that of other infectious diseases, shows marked species barrier, either being unable to infect heterologous species or difficult to form transmission experimentally. The similarity of the amino acid sequences of PrP among species is believed to be one of the elements in controlling the transmission TSE interspecies. Other factors, such as prion strains and host's microenvironment, may also participate in the process. METHODS: Two mouse-adapted strains 139A and ME7 were cerebrally inoculated to Golden hamsters. Presences of scrapie associate fibril (SAF) and PrPSc in brains of the infected animals were tested by TEM assays and Western blots dynamically during the incubation periods. The pathogenic features of the novel prions in hamsters, including electrophoretic patterns, glycosylating profiles, immunoreactivities, proteinase K-resistances and conformational stabilities were comparatively evaluated. TSE-related neuropathological changes were assayed by histological examinations. RESULTS: After long incubation times, mouse-adapted agents 139A and ME7 induced experimental scrapie in hamsters, respectively, showing obvious spongiform degeneration and PrPSc deposits in brains, especially in cortex regions. SAF and PrPSc in brains were observed much earlier than the onset of clinical symptoms. The molecular characteristics of the newly-formed PrPSc in hamsters, 139A-ha and ME7-ha, were obviously distinct from the original mouse agents, however, greatly similar as that of a hamster-adapted scrapie strain 263 K. Although the incubation times and main disease signs of the hamsters of 139A-ha and ME7-ha were different, the pathogenic characteristics and neuropathological changes were highly similar. CONCLUSIONS: This finding concludes that mouse-adapted agents 139A and ME7 change their pathogenic characteristics during the transmission to hamsters. The novel prions in hamsters' brains obtain new molecular properties with hamster-specificity.

Diphenylpyrazole-derived compounds increase survival time of mice after prion infection.

Transmissible spongiform encephalopathies (TSEs) represent a group of fatal neurodegenerative disorders that can be transmitted by natural infection or inoculation. TSEs include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, and Creutzfeldt-Jakob disease (CJD) in humans. The emergence of a variant form of CJD (vCJD), which has been associated with BSE, produced strong pressure to search for effective treatments with new drugs. Up to now, however, TSEs have proved incurable, although many efforts have been made both in vitro and in vivo to search for potent therapeutic and prophylactic compounds. For this purpose, we analyzed a compound library consisting of 10,000 compounds with a cell-based high-throughput screening assay dealing with scrapie-infected scrapie mouse brain and ScN(2)A cells and identified a new class of inhibitors consisting of 3,5-diphenylpyrazole (DPP) derivatives. The most effective DPP derivative showed half-maximal inhibition of PrP(Sc) formation at concentrations (IC(50)) of 0.6 and 1.2 µM, respectively. This compound was subsequently subjected to a number of animal experiments using scrapie-infected wild-type C57BL/6 and transgenic Tga20 mice. The DPP derivative induced a significant increase of incubation time both in therapeutic and prophylactic experiments. The onset of the prion disease was delayed by 37 days after intraperitoneal and 42 days after oral application, respectively. In summary, we demonstrate a high in vitro efficiency of DPP derivatives against prion infections that was substantiated in vivo for one of these compounds. These results indicate that the novel class of DPP compounds should comprise excellent candidates for future therapeutic studies.

The evaluation of exposure risks for natural transmission of scrapie within an infected flock.

BACKGROUND: Although the epidemiology of scrapie has been broadly understood for many years, attempts to introduce voluntary or compulsory controls to eradicate the disease have frequently failed. Lack of precision in defining the risk factors on farm has been one of the challenges to designing control strategies. This study attempted to define which parts of the annual flock management cycle represented the greatest risk of infection to naive lambs exposed to the farm environment at different times. RESULTS: In VRQ/VRQ lambs exposed to infected sheep at pasture or during lambing, and exposed to the buildings in which lambing took place, the attack rate was high and survival times were short. Where exposure was to pasture alone the number of sheep affected in each experimental group was reduced, and survival times were longer and related to length of exposure. CONCLUSION: At the flock level, eradication and control strategies for scrapie must take into account the need to decontaminate buildings used for lambing, and to reduce (or prevent) the exposure of lambs to infected sheep, especially in the later stages of incubation, and at lambing. The potential for environmental contamination from pasture should also be considered. Genotype selection may still prove to be the only viable tool to prevent infection from contaminated pasture, reduce environmental contamination and limit direct transmission from sheep to sheep.

Reduction of prion infectivity and levels of scrapie prion protein by lithium aluminum hydride: implications for RNA in prion diseases.

Previous studies indicate that RNA may be required for proteinase-resistant prion protein (PrP) amplification and for infectious prion formation in vitro, suggesting that RNA molecules may function as cellular cofactors for abnormal PrP (PrPSc) formation and become part of the structure of the infectious agent. To address this question, we used chemicals that can cleave phosphodiester bonds of RNA and assessed their effects on the infectious agent. Lithium aluminum hydride, a reducing agent that can induce reductive cleavage of oxidized molecules such as carbonyls, carboxyl acids, esters, and phosphodiester bonds, did not affect cellular PrP degradation; however, it destroyed PrPSc, extended the scrapie incubation period, and markedly reduced total RNA concentrations. These results prompted us to investigate whether RNA molecules are cofactors for PrPSc propagation. RNase A treatment of partially purified PrP and of 263K scrapie brain homogenates was sufficient to increase the sensitivity of PrPSc to proteinase K degradation. This is the first evidence that suggests that RNA molecules are a component of PrPSc. Treatment with RNase A alone and PrP degradation by RNase A plus proteinase K in vitro, however, did not result in loss of scrapie infectivity compared with the effects of lithium aluminum hydride. Together, these data suggest that RNA molecules may be important for maintaining the structure of PrPSc and that oxidized molecules can be important in scrapie agent replication and prion infectivity.

Quantifying diversity losses due to selection for scrapie resistance in three endangered Spanish sheep breeds using microsatellite information.

The effect of selection for scrapie resistance on genetic variability in three endangered Spanish sheep breeds (Colmenareña, Mallorquina and Rubia de El Molar) was studied using two different criteria for quantifying contributions to genetic variability: (a) molecular coancestry or genetic identity; and (b) average number of alleles per locus or allelic richness. A total of 236 (81 Colmenareña, 76 Mallorquina and 79 Rubia de El Molar) individuals were genotyped for the PrP gene and for 22 microsatellite markers. The analyses assumed a selective policy aimed at the elimination of the VRQ allele and the reduction of the frequency of the ARQ/ARQ genotype. These goals are approached by rejecting for breeding those individuals with the highest susceptibility for scrapie (risk groups R4 and R5) in a genetic scenario with no previous selection programmes considering the PrP gene polymorphism carried out. When all the individuals classified into risk groups R4 and R5 were removed from the dataset, the total molecular coancestry slightly increased in the Colmenareña breed illustrating that the carriers of undesirable PrP genotypes are not essential to maintain its overall gene diversity. When the allelic richness was considered, the removal of the R4 and R5 individuals gave high losses in the Rubia de El Molar breed. The analyses carried out considering the sex of the individuals informed that most increases in genetic identity in the Colmenareña breed resulted from the removal of the R4 and R5 males while in the Mallorquina breed resulted from the removal of the undesirable females. Losses of diversity in the Rubia de El Molar breed were basically independent of the sex of the individuals due to the balanced contributions to diversity of both sexes. As a general recommendation, not all the individuals of undesirable risk groups should be rejected for reproduction at the same time to avoid irretrievable loses of genetic diversity but according to the sex of the individuals.

Role of copper and manganese in prion disease progression.

The cellular prion protein (PrP(C)), a copper binding protein has a primary role in the pathogenesis of in prion diseases. In these diseases alterations in the levels of copper and manganese have been described but how these alterations are involved in the pathogenesis is still unknown. Here we analysed synaptosomes of scrapie infected mice and observed a significant reduction in the amount of copper and an increase of the manganese content at day 100 after infection. Moreover a reduction of the copper content in mouse brains induced by application of copper poor diets was found to reduce the survival time of scrapie infected mice significantly, whereas enhanced administration of copper induced a significant delay in prion disease onset. Interestingly a significant higher amount of PrP(C) full length and misfolded PK-resistant PrP was observed in mice that were treated with an enhanced copper diet compared to controls. Moreover we could demonstrate that in healthy mock infected mice, a Cu(2+) rich/Mn(2+) poor diet induced a significantly decreased cleavage capability of PrP(C) compared to control mice. These new findings suggest that the copper content in mouse brains exerts an influence on the amount of PrP(C) and its cleavage properties and may affect the PrP conversion by depleted availability of functional PrP full length.

Alkaline hydrolysis of mouse-adapted scrapie for inactivation and disposal of prion-positive material.

Prion diseases such as bovine spongiform encephalopathy, chronic wasting disease, and scrapie pose serious risks to human and animal health due to a host of disease-specific factors, including the resistance of infectious prions (PrP(Sc)) to natural degradation and to most commercial inactivation procedures. In an attempt to address this concern, a mouse model was used to compare the efficacy of an alkaline hydrolysis process with a simulated continuous-flow rendering treatment for disposal of PrP(Sc)-infected biological material. Female C57/BL6 mice (N = 120) were randomly divided into 4 treatment groups (n = 30), and each mouse was injected intraperitoneally with their designated treatment inoculum. Treatment groups 1 and 2 served as the positive and negative controls, respectively. Group 3 was inoculated with rendered scrapie-positive mouse brain material to investigate the effectiveness of simulated continuous-flow rendering practices to reduce or eliminate PrP(Sc). Group 4 was inoculated with hydrolyzed scrapie-positive mouse brain material to determine the sterilizing effect of alkaline hydrolysis on PrP(Sc). Mice were monitored for overt signs of disease, and those showing clinical signs were killed to prevent undue suffering. Brains were obtained from all mice that died (or were killed) and analyzed with an ELISA for the presence of PrP(Sc). Results indicated that the simulated continuous-flow rendering treatment used for preparing the rendering treatment group inoculum failed to completely eliminate PrP(Sc). Rendering delayed, but did not stop, clinical mouse-adapted scrapie transmission. Compared with positive controls, the rendering treatment group experienced an approximate 45-d average delay in days to death (250 vs. 205 d for positive controls; P < 0.0001) and a death loss of 73.9% (P = 0.0094). Positive controls suffered 100% death loss. The results validated the efficacy of the alkaline hydrolysis treatment to inactivate all PrP(Sc) because no alkaline hydrolysis treatment group mice succumbed to the disease (P < 0.0001). Based on our results, alkaline hydrolysis should be considered by the animal rendering and beef packing industries as an alternative to incineration, landfill burial, and rendering for disposing of biological material potentially infected or contaminated with prion disease.

The bank vole (Myodes glareolus) as a sensitive bioassay for sheep scrapie.

Despite intensive studies on sheep scrapie, a number of questions remain unanswered, such as the natural mode of transmission and the amount of infectivity which accumulates in edible tissues at different stages of scrapie infection. Studies using the mouse model proved to be useful for recognizing scrapie strain diversity, but the low sensitivity of mice to some natural scrapie isolates hampered further investigations. To investigate the sensitivity of bank voles (Myodes glareolus) to scrapie, we performed end-point titrations from two unrelated scrapie sources. Similar titres [10(5.5) ID50 U g(-1) and 10(5.8) ID50 U g(-1), both intracerebrally (i.c.)] were obtained, showing that voles can detect infectivity up to 3-4 orders of magnitude lower when compared with laboratory mice. We further investigated the relationships between PrPSc molecular characteristics, strain and prion titre in the brain and tonsil of the same scrapie-affected sheep. We found that protease-resistant PrPSc fragments (PrPres) from brain and tonsil had different molecular features, but induced identical disease phenotypes in voles. The infectivity titre of the tonsil estimated by incubation time assay was 10(4.8) i.c. ID50 U g(-1), i.e. fivefold less than the brain. This compared well with the relative PrPres content, which was 8.8-fold less in tonsil than in brain. Our results suggest that brain and tonsil harboured the same prion strain showing different glycoprofiles in relation to the different cellular/tissue types in which it replicated, and that a PrPSc-based estimate of scrapie infectivity in sheep tissues could be achieved by combining sensitive PrPres detection methods and bioassay in voles.

Scrapie resistance in ARQ sheep.

Variation in the ovine prion protein amino acid sequence influences scrapie progression, with sheep homozygous for A(136)R(154)Q(171) considered susceptible. This study examined the association of survival time of scrapie-exposed ARQ sheep with variation elsewhere in the ovine prion gene. Four single nucleotide polymorphism alleles were associated with prolonged survival. One nonsynonymous allele (T112) was associated with an additional 687 days of survival for scrapie-exposed sheep compared to M112 sheep (odds ratio, 42.5; P = 0.00014). The only two sheep homozygous for T112 (TARQ) did not develop scrapie, suggesting that the allelic effect may be additive. These results provide evidence that TARQ sheep are genetically resistant to development of classical scrapie.