Natural Scrapie Cases Research Articles

Mixtures of prion substrains in natural scrapie cases revealed by ovinised murine models

Phenotypic variability in prion diseases, such as scrapie, is associated to the existence of prion strains, which are different pathogenic prion protein (PrPSc) conformations with distinct pathobiological properties. To faithfully study scrapie strain variability in natural sheep isolates, transgenic mice expressing sheep cellular prion protein (PrPC) are used. In this study, we used two of such models to bioassay 20 scrapie isolates from the Spain-France-Andorra transboundary territory. Animals were intracerebrally inoculated and survival periods, proteinase K-resistant PrP (PrPres) banding patterns, lesion profiles and PrPSc distribution were studied. Inocula showed a remarkable homogeneity on banding patterns, all of them but one showing 19-kDa PrPres. However, a number of isolates caused accumulation of 21-kDa PrPres in TgShp XI. A different subgroup of isolates caused long survival periods and presence of 21-kDa PrPres in Tg338 mice. It seemed that one major 19-kDa prion isoform and two distinct 21-kDa variants coexisted in source inocula, and that they could be separated by bioassay in each transgenic model. The reason why each model favours a specific component of the mixture is unknown, although PrPC expression level may play a role. Our results indicate that coinfection with more than one substrain is more frequent than infection with a single component.

Nonpathogenic Heterologous Prions Can Interfere with Prion Infection in a Strain-Dependent Manner.

Co-occurrence of different prion strains into the same host has been recognized as a natural phenomenon for several sporadic Creutzfeldt-Jakob disease (sCJD) patients and natural scrapie cases. The final outcome of prion coinfection is not easily predictable. In addition to the usual factors that influence prion conversion, the replication of one strain may entail positive or negative consequences to the other. The main aim of this study was to gain insights into the prion coinfection and interference concepts and their potential therapeutic implications. Here, different mouse models were challenged with several combinations of prion strains coupled on the basis of the lengths of their incubation periods and the existence/absence of a species barrier in the tested animal model. We found that nontransmissible strains can interfere the replication of fully transmissible strains when there is a species transmission barrier involved, as happened with the combination of a mouse (22L) and a human (sCJD) strain. However, this phenomenon seems to be strain dependent, since no interference was observed when the human strain coinoculated was vCJD. For the other combinations tested in this study, the results suggest that both strains replicate independently without effect on the replication of one over the other. It is common that the strain with more favorable conditions (e.g., a higher speed of disease development or the absence of a species barrier) ends being the only one detectable at the terminal stage of the disease. However, this does not exclude the replication of the least favored strain, leading to situations of the coexistence of prion strains.IMPORTANCE As a general conclusion, the outcome of prion coinfection is strongly dependent on the strain combination and the model utilized and is therefore difficult to predict. The coexistence of several prion strains may remain undetected if one of the strains has more favorable conditions to replicate in the host. The use of several models (such as a transgenic mouse expressing PrP from different species) to analyze field prion isolates is recommended to avoid this situation. The inference effect exerted by nonreplicative prion strains should be considered an interesting tool to advance in new therapeutic strategies for treating prion diseases; it may even be a proper therapeutic strategy.

Neuropathological changes correlate temporally but not spatially with selected neuromodulatory responses in natural scrapie

Neuropathological changes classically associated with sheep scrapie do not always correlate with clinical disease. We aimed to determine if selected neuromodulatory responses were altered during the course of the infection as it has been described in Creutzfeldt-Jakob disease and experimental bovine spongiform encephalopathy. Hemi-brains from healthy sheep and natural scrapie cases at two stages of infection were examined for biochemical alterations related to the expression of type I metabotropic glutamatergic receptors (mGluR(1) ) and type I adenosine receptors I (A(1) R), and of selected downstream intermediate signalling targets. Immunohistochemistry for different scrapie-related neuropathological changes was performed in the contralateral hemi-brains. PrP(d) deposition, spongiform change, astrocytosis and parvalbumin expression were significantly altered in brains from clinically affected sheep compared with preclinical cases and negative controls; the latter also showed significantly higher immunoreactivity for synaptophysin than clinical cases. Between clinically affected and healthy sheep, no differences were found in the protein levels of mGluR(1) , while phospholipase Cβ1 expression in terminally ill sheep was increased in some brain areas but decreased in others. Adenyl cyclase 1 and A(1) R levels were significantly lower in various brain areas of affected sheep. No abnormal biochemical expression levels of these markers were found in preclinically infected sheep. These findings point towards an involvement of mGluR(1) and A(1) R downstream pathways in natural scrapie. While classical prion disease lesions and neuromodulatory responses converge in some affected regions, they do not do so in others suggesting that there are independent regulatory factors for distinct degenerative and neuroprotective responses.

Histopathological Studies of “CH1641-Like” Scrapie Sources Versus Classical Scrapie and BSE Transmitted to Ovine Transgenic Mice (TgOvPrP4)

The possibility of the agent causing bovine spongiform encephalopathy (BSE) infecting small ruminants is of serious concern for human health. Among scrapie cases, the CH1641 source in particular appears to have certain biochemical properties similar to the BSE strain. In France, several natural scrapie cases were identified as “CH1641-like” natural scrapie isolates in sheep and goats. The Tg(OvPrP4) mouse line expressing the ovine prion protein is a sensitive model for studying and identifying strains of agents responsible for scrapie and BSE. This model is also very useful when studying specific scrapie source CH1641, known to be not transmissible to wild-type mice despite the similarity of some of its biochemical properties to those of the BSE strain. As it is important to be able to fully distinguish CH1641 from BSE, we herein report the histopathological data from CH1641 scrapie transmission experiments compared to specific cases of “CH1641-like” natural scrapie isolates in sheep, murine scrapie strains and BSE. In addition to the conventional vacuolar lesion profile approach and PrPd brain mappings, an innovative differential PET-blot analysis was introduced to classify the different strains of agent and revealed the first direct concordance between ways of grouping strains on the basis of PrPd biochemical characteristics.

Classic Scrapie in Sheep with the ARR/ARR Prion Genotype in Germany and France

In the past, natural scrapie and bovine spongiform encephalopathy (BSE) infections have essentially not been diagnosed in sheep homozygous for the A136R154R171 haplotype of the prion protein. This genotype was therefore assumed to confer resistance to BSE and classic scrapie under natural exposure conditions. Hence, to exclude prions from the human food chain, massive breeding efforts have been undertaken in the European Union to amplify this gene. We report the identification of 2 natural scrapie cases in ARR/ARR sheep that have biochemical and transmission characteristics similar to cases of classic scrapie, although the abnormally folded prion protein (PrP(Sc)) was associated with a lower proteinase-K resistance. PrP(Sc) was clearly distinct from BSE prions passaged in sheep and from atypical scrapie prions. These findings strongly support the idea that scrapie prions are a mosaic of agents, which harbor different biologic properties, rather than a unique entity.

Immunohistochemistry for PrPSc in natural scrapie reveals patterns which are associated with the PrP genotype

Immunohistochemistry for PrPSc is used widely in scrapie diagnosis. In natural scrapie cases the use of immunohistochemistry (IHC) has revealed the existence of up to 12 different morphological types of immunostained deposits. The significance of this pattern variability in relation to genotype has not been studied extensively in natural disease. In this study we recorded in detail PrPSc patterns at the obex level of the medulla oblongata from 163 animals derived from 55 flocks which presented through passive surveillance in the UK and Italy. A strong association was seen between PrPSc patterns and PrP genotype, particularly in relation to codon 136. In a blind assessment of this association we were able to predict, with over 80% accuracy, the genotype of 151 scrapie cases which were presented through passive surveillance from 13 farms. The genotype of these cases was ARQ/ARQ or VRQ/VRQ. The association of PrPsc patterns with genotype was generally stronger in those farms where all the affected animals belonged to a single genotype compared with farms where both genotypes were identified, with the exception of one farm in which the genotype of all affected sheep was ARQ/ARQ and the PrPSc patterns were of the VRQ/VRQ type. Our observations support the hypothesis that the observed association between specific IHC patterns and genotypes may in fact be strain driven but in natural disease individual scrapie strains may demonstrate a genotypic tropism.

Proteinase K-sensitive disease-associated ovine prion protein revealed by conformation-dependent immunoassay

PrPSc [abnormal disease-specific conformation of PrP (prion-related protein)] accumulates in prion-affected individuals in the form of amorphous aggregates. Limited proteolysis of PrPSc results in a protease-resistant core of PrPSc of molecular mass of 27-30 kDa (PrP27-30). Aggregated forms of PrP co-purify with prion infectivity, although infectivity does not always correlate with the presence of PrP27-30. This suggests that discrimination between PrPC (normal cellular PrP) and PrPSc by proteolysis may underestimate the repertoire and quantity of PrPSc subtypes. We have developed a CDI (conformation-dependent immunoassay) utilizing time-resolved fluorescence to study the conformers of disease-associated PrP in natural cases of sheep scrapie, without using PK (proteinase K) treatment to discriminate between PrPC and PrPSc. The capture-detector CDI utilizes N-terminal- and C-terminal-specific anti-PrP monoclonal antibodies that recognize regions of the prion protein differentially buried or exposed depending on the extent of denaturation of the molecule. PrPSc was precipitated from scrapie-infected brain stem and cerebellum tissue following sarkosyl extraction, with or without the use of sodium phosphotungstic acid, and native and denatured PrPSc detected by CDI. PrPSc was detectable in brain tissue from homozygous VRQ (V136 R154 Q171) and ARQ (A136 R154 Q171) scrapie-infected sheep brains. The highest levels of PrPSc were found in homozygous VRQ scrapie-infected brains. The quantity of PrPSc was significantly reduced, up to 90% in some cases, when samples were treated with PK prior to the CDI. Collectively, our results show that the level of PrPSc in brain samples from cases of natural scrapie display genotypic differences and that a significant amount of this material is PK-sensitive.

Prion protein (PrP) gene polymorphisms associated with natural scrapie cases and their flock-mates in Ireland

The PrP genotypes associated with natural scrapie in Ireland were determined and a comparison was made between genotypes found in scrapie-infected sheep and those found in healthy animals from scrapie-infected flocks. Seven PrP genotypes were identified in scrapie-infected animals: VV 136 RR 154 QQ 171, VA 136 RR 154 QQ 171, VA 136 RR 154 QR 171, VA 136 RR 154 QH 171, AA 136 RR 154 QQ 171, AA 136 RR 154 QH 171 and AA 136RR 154HH 171. Of 11 scrapie-infected flocks, 15 genotypes were identified in the healthy flock-mates. The genotypes identified in scrapie-affected animals were also all identified in healthy flock-mates. In 9 of the 11 flocks studied, the genotype frequencies among scrapie-infected animals were significantly different from those among healthy flock-mates. The results show that there is a significant risk of developing the clinical signs of scrapie associated with particular PrP genotypes in the Irish sheep population. The association between the V 136R 154Q 171 allele and scrapie was evident, as was the association between A 136R 154R 171 and resistance to developing the clinical signs of scrapie. The presence of the A 136H 154Q 171 allele in the flocks examined resulted in a decreased risk of developing scrapie compared to the presence of the A 136R 154Q 171.

Distribution and accumulation of PrP in gut-associated and peripheral lymphoid tissue of scrapie-affected Suffolk sheep.

The distribution of disease-associated prion protein (PrP) was investigated in eight animals (20-24 months of age) from a flock of Suffolk sheep that had experienced frequent cases of natural scrapie over a period of several years. Tissue from the central nervous system (CNS), alimentary tract, peripheral nervous system and lymphoreticular system was examined by histopathology and immunohistochemistry. The lymphoid tissues were subjected further to histoblot and immunofluorescence examination. The four clinically affected PrP(ARQ/ARQ) sheep had widespread accumulations of disease-associated PrP in the CNS, lymphoreticular system and peripheral ganglia. In the two PrP(ARQ/ARQ) sheep that did not show clinical signs of scrapie, only limited vacuolation and PrP accumulation were detected in the brain, but the results from the lymphoreticular system and peripheral nervous system were comparable with the clinically affected animals. The remaining PrP(ARR/ARR) and PrP(ARR/ARQ) sheep did not show proteinase K-resistant PrP accumulations in the lymphoid tissues examined and immunohistochemistry did not reveal the presence of disease-associated PrP. In lymphoid tissues of the PrP(ARQ/ARQ) sheep, the dominant localization of disease-associated PrP was in lymphoid nodules and double immunofluorescence labelling for PrP and CD21 provided further support for the role of follicular dendritic cells in scrapie in sheep. A striking finding in the present study was the large accumulations of disease-associated PrP in the lymphoid nodules of the alimentary tract at the late sub-clinical and clinical stage of the infection. The study also identified disease-associated PrP in extra-nodular sites of lymphoid tissues, such as the marginal zone of the spleen, and these observations were used to argue that cells of the mononuclear phagocyte system of sheep may be involved in the uptake, transport, elimination and shedding of the scrapie agent.

Comparison of French natural scrapie isolates with bovine spongiform encephalopathy and experimental scrapie infected sheep

We compared the glycoform pattern of the abnormal prion protein (PrP Sc) detected by immunoblotting in 21 sheep with natural scrapie, from 21 different outbreaks identified in France since 1996, with a bovine spongiform encephalopathy (BSE)-infected sheep. All the natural scrapie isolates had a higher molecular mass of the unglycosylated PrP Sc than in BSE-infected sheep. In the latter case, this molecular mass appeared identical to that found in the CH 1641 experimental scrapie strain (type C pattern), whereas in natural scrapie cases it was similar to that found in the SSBP/1 experimental scrapie strains. These results suggest that all French natural scrapie isolates studied so far would belong, as SSBP/1, to the group of scrapie cases with type A electrophoretic pattern.

Prion protein gene polymorphisms in sheep with natural scrapie and healthy controls in Norway.

Two-hundred and forty healthy sheep and 32 cases of natural scrapie in Norway were analysed for disease-linked polymorphisms in the prion protein (PrP) gene. Scrapie was strongly associated with the presence of a valine polymorphism at codon 136 (V136), as 68.8% of the cases were homozygous (VV136) and 15.6% were valine/alanine heterozygous (VA136). All cases were homozygous arginine/arginine at codon 154 (RR154), except two which were homozygous histidine/histidine (HH154). All cases except two were homozygous glutamine/glutamine at codon 171 (QQ171), the two exceptions being heterozygous glutamine/arginine (QR171). More than 80% of all scrapie cases in Norway have occurred in a Cheviot-related crossbred type of sheep called Rygja. This type of sheep, which is largely restricted to the south-western coast, carries the V136 allele at a higher frequency than do other breeds of Norwegian sheep. Polymorphisms at codons 138 and 151 are also described.

The distribution of scrapie-associated fibrils in neural and non-neural tissues of advanced clinical cases of natural scrapie in sheep

The distribution of scrapie-associated fibrils (SAFs) throughout four brain regions, the pituitary gland, along the whole length of the spinal cord and in the sciatic nerve was assessed in 10 sheep terminally affected by scrapie and in four control sheep. Tonsils, retropharyngeal, broncho-mediastinal and mesenteric lymph nodes, the distal ileum, proximal colon and spleen were also examined for fibrils in all 14 sheep. Fibrils were detected in all four brain regions and throughout the length of the spinal cord in nine of the scrapie affected sheep. SAFs were not detectable in any of the sciatic nerve samples tested. In one of the 10 clinically affected sheep only minimal lesions were found by histopathology and fibrils were detected only from the cerebrum and one spinal cord region (taken at the C1 C2 vertebrae). Fibrils were not detected in the tonsils or retropharyngeal lymph nodes but were detected in other non-neural tissues of some of the scrapie-affected sheep. These tissues included pituitary gland, broncho-mediastinal and mesenteric portal lymph nodes, distal ileum, proximal colon and spleen. Fibrils could not be detected in any of the tissues taken from the four control sheep.

Neuropathology of scrapie: a study of the distribution patterns of brain lesions in 222 cases of natural scrapie in sheep, 1982–1991

Detailed neuropathological findings in 222 cases of naturally occurring scrapie from Great Britain are described. The material consisted of formalin-fixed brain from eight breeds of sheep submitted between 1982 and...

PrP Polymorphisms Associated with Natural Scrapie Discovered by Denaturing Gradient Gel Electrophoresis

Scrapie is a transmissible degenerative disease of the central nervous system occurring naturally in sheep and goats. An abnormal protease-resistant form of the host-encoded prion protein (PrP) accumulates in the brains of affected animals. As Sip, a gene controlling the incubation period of experimental and natural scrapie, is linked to the single-copy sheep PrP gene, we sought PrP coding sequence polymorphisms in flocks from the Romanov and Ile-de-France breeds endemically affected with natural scrapie. DNA samples from 153 sheep, including 29 natural scrapie cases, were screened by using polymerase chain reactions and denaturing gradient gel electrophoresis. Four predicted amino acid substitutions were found in the center of the PrP coding region: 112 Met-->Thr, 136 Ala-->Val, 154 Arg-->His, and 171 Gln-->Arg. These substitutions appeared mutually exclusive, defining five coding alleles. The 136Val allele, substituting a highly conserved Ala residue, in a homozygous or heterozygous state correlated with susceptibility to natural scrapie (chi 2 = 64.33, P < 0.001). This correlation indicates that the 136 Val allele may modulate development of the disease, implying a pivotal role for PrP molecules in natural scrapie, as has been observed for experimental scrapie and human prion diseases.