Spongiform Encephalopathy In Sheep Research Articles

Genetic variability of the prion protein gene in Indonesian goat breeds

Scrapie is a naturally occurring transmissible spongiform encephalopathy in sheep and goats. Resistance or susceptibility of small ruminants to classical scrapie is influenced by polymorphisms in the prion protein gene (PRNP). PRNP variability in Indonesian indigenous goat breeds has not been investigated so far and therefore was the goal of this study. Sanger sequencing of the PRNP gene coding region in 72 goats of the seven Indonesian breeds Kacang, Gembrong, Samosir, Kejobong, Benggala, Jawarandu, and Peranakan Etawah revealed three amino acid substitutions, namely W102G, H143R, and S240P. Some silent mutations were also found at codons 42 (a/g), 138 (c/t), and 179 (g/t). The PRNP alleles K222 and D/S146 known to have significant protective effects on resistance to classical scrapie in goats were not detected. The allele R143, which may have a moderate protective effect, had a frequency of 12% among the analyzed Indonesian goat breeds. While R143 was missing in Kacang and Benggala, its frequency was highest in the breed Gembrong (32%). No scrapie cases have been reported in Indonesia until now. However, in the case that selection for protective PRNP variants would become a breeding goal, the analyzed breeds will not be very useful resources. Other goat breeds which are present in the country should be investigated regarding resistance to scrapie, too.

Source genotype influence on cross species transmission of transmissible spongiform encephalopathies evaluated by RT-QuIC.

Scrapie is a naturally occurring transmissible spongiform encephalopathy of sheep and goats. This fatal neurodegenerative disease is caused by misfolding of the cellular prion protein to pathogenic β-rich conformers (PrPSc) that accumulate in higher order structures of the brain and other tissues. This conversion has been used for in vitro assays including serial protein misfolding amplification and real-time quaking induced conversion (RT-QuIC). RT-QuIC can be used for the detection of prions and for strain discrimination in a variety of biological tissues from humans and animals. In this study, we evaluated how PrPSc isolated from sheep of different genotypes after inoculation with the scrapie agent influence the fibril formation in vitro using RT-QuIC. We found that reaction mixtures seeded with PrPSc from genotype VRQ/VRQ sheep brains have better conversion efficiency with 132M elk substrate compared to reactions seeded with PrPSc from the brains of sheep with the ARQ/ARQ genotype no matter which strain of scrapie was used to seed the reactions. We also inoculated transgenic mice expressing 132M elk PRNP (Tg12) with the scrapie agent from different genotypes of sheep to compare with our RT-QuIC results. The bioassays support the data showing a significantly shorter incubation period for inoculum from VRQ/VRQ sheep when compared to inoculum from ARQ/ARQ sheep. Thus, we conclude that the genotype of both source and recipient can strongly influence transmission.

Modelling of strategies for genetic control of scrapie in sheep: The importance of population structure.

Scrapie is a transmissible spongiform encephalopathy in sheep and an example of a disease that may be controlled through breeding for disease resistance. Member states of the European Union have introduced strategies for breeding against scrapie based on the selection of genetically resistant breeding rams. An ambitious strategy adopted in The Netherlands consisted of selecting resistant rams for breeding throughout both breeding and production sectors. Mathematical modelling of the effect of a breeding program on the spreading capacity of scrapie in a national flock is needed for making assessments on how long a breeding strategy needs to be maintained to achieve disease control. Here we describe such a model applied to the Dutch situation, with the use of data on the genetic content of the Dutch sheep population as well as on scrapie occurrence in this population. We show that the time needed for obtaining scrapie control depends crucially on two parameters measuring sheep population structure: the between-flock heterogeneity in genotype frequencies, and the heterogeneity of mixing (contact rates) between sheep flocks. Estimating the first parameter from Dutch genetic survey data and assuming scenario values for the second one, enables model prediction of the time needed to achieve scrapie control in The Netherlands.

Real-time qPCR is a powerful assay to estimate the 171 R/Q alleles at the PrP locus directly in a flock's raw milk: A comparison with the targeted next-generation sequencing

The hazard to human health represented by transmissible spongiform encephalopathies in sheep is one of the major reasons for implementing the genetic selection plan to break down prion diseases. The problem is particularly important because of the risk of disease transmission from ewe to lamb via milk or colostrum. In order to establish an active and convenient monitoring of the flocks already undergone genetic selection and thus, indirectly increase consumers’ security, the challenge of the work was quantifying the classical scrapie risk in bulk milk. A new quantitative real-time PCR assay for the estimation of the 171 R and Q allelic frequencies in a DNA pool representative of all the lactating ewes present in a flock was optimized and validated “in field”. The repeatability range was 3.69–5.27 for R and 4.20–5.75 for Q. The ruggedness of the allele frequencies resulted 4.26 for R and 4.77 for Q. Regarding the validation “in field”, none of the considered sources of variability (flock, month, number of genotyped animals and somatic cell count) showed a significant effect on flock and milk at the linear model. The targeted next-generation sequencing was also tested to evaluate its applicability in this context. Results show that the real-time PCR assay could represent a valid tool for the determination of 171 R/Q allele frequencies in bulk milk. The implementation of a service for breeder self-control along the production chain would aim to increase the production of high-security dairy products, while monitoring over time of the effects of genetic selection in the flocks.

Selection of Distinct Strain Phenotypes in Mice Infected by Ovine Natural Scrapie Isolates Similar to CH1641 Experimental Scrapie

A few cases of transmissible spongiform encephalopathies in sheep have been described in France in which the protease-resistant prion protein (PrP(res)) exhibited some features in Western blot of experimental bovine spongiform encephalopathy in sheep. Their molecular characteristics were indistinguishable from those produced in the CH1641 experimental scrapie isolate. Four of these CH1641-like isolates were inoculated intracerebrally into wild-type C57Bl/6 mice. In striking contrast to previous results in ovine transgenic mice, CH1641 transmission in wild-type mice was efficient. Several components of the strain signature, that is, PrP(res) profile, brain distribution, and morphology of the deposits of the disease-associated prion protein, had some similarities with "classical" scrapie and clearly differed from both bovine spongiform encephalopathy in sheep and CH1641 transmission in ovine transgenic mice. These results on CH1641-like isolates in wild-type mice may be consistent with the presence in these isolates of mixed conformers with different abilities to propagate and mediate specific disease phenotypes in different species.

Paraffin-embedded tissue blot as a sensitive method for discrimination between classical scrapie and experimental bovine spongiform encephalopathy in sheep

The paraffin-embedded tissue (PET) blot was modified for use as a tool to differentiate between classical scrapie and experimental bovine spongiform encephalopathy (BSE) in sheep. Medulla (obex) from 21 cases of classical scrapie and 6 cases of experimental ovine BSE were used to develop the method such that it can be used as a tool to differentiate between BSE and scrapie in the same way that differential immunohistochemistry (IHC) has been used previously. The differential PET blot successfully differentiated between all of the scrapie and ovine BSE cases. Differentiation was permitted more easily with PET blot than by differential IHC, with accurate observations possible at the macroscopic level. At the microscopic level, sensitivity was such that discrimination by the differential PET blot could be made with more confidence than with differential IHC in cases where the immunohistochemical differences were subtle. The differential PET blot makes use of harsh epitope demasking conditions, and, because of the differences in the way prion protein is processed in different prion diseases, it can serve as a new, highly sensitive method to discriminate between classical scrapie and experimental BSE in sheep.

Breeding with resistant rams leads to rapid control of classical scrapie in affected sheep flocks

Susceptibility to scrapie, a transmissible spongiform encephalopathy in sheep, is modulated by the genetic make-up of the sheep. Scrapie control policies, based on selecting animals of resistant genotype for breeding, have recently been adopted by the Netherlands and other European countries. Here we assess the effectiveness of a breeding programme based on selecting rams of resistant genotype to obtain outbreak control in classical scrapie-affected sheep flocks under field conditions. In six commercially-run flocks following this breeding strategy, we used genotyping to monitor the genotype distribution, and tonsil biopsies and post-mortem analyses to monitor the occurrence of scrapie infection. The farmers were not informed about the monitoring results until the end of the study period of six years. We used a mathematical model of scrapie transmission to analyze the monitoring data and found that where the breeding scheme was consistently applied, outbreak control was obtained after at most four years. Our results also show that classical scrapie control can be obtained before the frequency of non-resistant animals is reduced to zero in the flock. This suggests that control at the national scale can be obtained without a loss of genetic polymorphisms from any of the sheep breeds.

The role of mathematical modelling in understanding the epidemiology and control of sheep transmissible spongiform encephalopathies: a review

To deal with the incompleteness of observations and disentangle the complexities of transmission much use has been made of mathematical modelling when investigating the epidemiology of sheep transmissible spongiform encephalopathies (TSE) and, in particular, scrapie. Importantly, these modelling approaches allow the incidence of clinical disease to be related to the underlying prevalence of infection, thereby overcoming one of the major difficulties when studying these diseases. Models have been used to investigate the epidemiology of scrapie within individual flocks and at a regional level; to assess the efficacy of different control strategies, especially selective breeding programmes based on prion protein (PrP) genotype; to interpret the results of scrapie surveillance; and to inform the design of surveillance programmes. Furthermore, mathematical modelling has played an important role when assessing the risk to human health posed by the possible presence of bovine spongiform encephalopathy in sheep. Here, we review the various approaches that have been taken when developing and analysing mathematical models for the epidemiology and control of sheep TSE and assess their impact on our understanding of these diseases. We also identify areas that require further work, discuss future challenges and identify data gaps.

Three serial passages of bovine spongiform encephalopathy in sheep do not significantly affect discriminatory test results

During the 1980s, bovine spongiform encephalopathy (BSE)-contaminated meat and bonemeal were probably fed to sheep, raising concerns that BSE may have been transmitted to sheep in the UK. The human disease, variant Creutzfeldt-Jakob disease, arose during the BSE epidemic, and oral exposure of humans to BSE-infected tissues has been implicated in its aetiology. The concern is that sheep BSE could provide another source of BSE exposure to humans via sheep products. Two immunological techniques, Western immunoblotting (WB) and immunohistochemistry (IHC), have been developed to distinguish scrapie from cases of experimental sheep BSE by the characteristics of their respective abnormal, disease-associated prion proteins (PrP(d)). This study compares the WB and IHC characteristics of PrP(d) from brains of primary, secondary and tertiary experimental ovine BSE cases with those of cattle BSE and natural sheep scrapie. Discrimination between experimental sheep BSE and scrapie remained possible by both methods, regardless of the route of challenge.

Neuroanatomical distribution of abnormal prion protein in naturally occurring atypical scrapie cases in Great Britain

Scrapie belongs to a group of diseases known as the transmissible spongiform encephalopathies or prion diseases. Two different categories of naturally occurring scrapie have been identified: classical scrapie, which was first recorded around 1750, and atypical scrapie or 'Nor-98', which was first identified in Norway in 1998. The molecular characteristics of atypical scrapie have been well defined, but detailed descriptions of the neuropathological phenotype are rare since the majority of cases have been detected through active surveillance programmes where only brainstem and cerebellum are collected for statutory diagnosis. In order to characterise the neuropathology of naturally occurring atypical scrapie in sheep, we examined multiple brain levels from 15 whole brains from field cases of atypical scrapie, both clinical suspects and fallen stock, collected in Great Britain between 2004 and 2006. We found that the distribution of disease-associated prion protein (PrP(Sc)) and vacuolation in atypical scrapie cases are very different to both classical scrapie and experimental bovine spongiform encephalopathy in sheep. Immunolabelling for PrP(Sc) is mild and restricted at the obex and more intense and widespread rostrally, particularly in the cerebellum, substantia nigra, thalamus and basal nuclei. Intracellular immunolabelling types are not seen, but distinctive white matter immunolabelling is widespread. Vacuolation associated with PrP(Sc) deposits was not observed in the brainstem neuroanatomical areas commonly affected in classical scrapie and bovine spongiform encephalopathy, but was instead most prominent in the cerebellar cortex and neocortex. This is the largest comprehensive descriptive study of atypical scrapie pathology to date, and provides baseline data against which other natural or experimental cases can be compared. It also reinforces the current recommendation to collect cerebellum in addition to brainstem to enable confident confirmation of this distinct disease phenotype within surveillance programmes.

TSE risk assessment from carcasses of ovine and caprine animals below 6 months of age from TSE infected flocks intended for human consumption - Scientific Opinion of the Panel on Biological Hazards.

TSE risk assessment from carcasses of ovine and caprine animals below 6 months of age from TSE infected flocks intended for human consumption - Scientific Opinion of the Panel on Biological Hazards.

The evidence of associations between prion protein genotype and production, reproduction, and health traits in sheep

The EU Commission issued a regulation in 2003, which requires all member states to implement a breeding programme for resistance to transmissible spongiform encephalopathies in sheep by selecting for specific alleles of the prion protein (PrP) gene. A key concern with regard to this regulation was that the intensive selection programmes, designed to increase resistance to scrapie, may have a negative impact on a range of other economically important production, reproduction, and disease traits in sheep. Such problems could arise for a number of reasons. Firstly, a number of breeds have a low frequency of the resistant PrP allele. Secondly, there may be a negative association between the resistant allele and animal performance. Thirdly, selection for scrapie resistance may reduce the rate of improvement towards current breeding goals. The evidence concerning the relationship between PrP genotype and reproduction, production, and disease traits is the subject of this review. We conclude that there is no evidence for a negative association between PrP genotype and reproduction traits (e.g. litter size), lamb performance traits (e.g. growth rate, conformation, carcass composition) or milk production. There is, however, a distinct paucity of information on the relationship between the PrP gene and disease traits. In this context it is noted that there are a number of genes located on chromosome 13, in close proximity to the PrP gene, that are involved in intracellular cell signalling, apoptosis, phagocytosis, and immune function. Thus further direct studies of key disease traits associated with sheep production systems are warranted.

Structural and functional analysis of the HSP90AA1 gene: distribution of polymorphisms among sheep with different responses to scrapie

Scrapie is a transmissible spongiform encephalopathy in sheep and goats. Susceptibility to this neurodegenerative disease is mainly controlled by point mutations at the PRNP locus. Other genes, apart from PRNP, have been reported to modulate resistance/susceptibility to scrapie. On the basis of several studies in Alzheimer and different transmissible spongiform encephalopathy models, HSP90AA1 was chosen as a putative positional and functional candidate gene that might be involved in the polygenic variance mentioned above. In the present work, the ovine HSP90AA1 gene including the promoter and other regulatory regions has been isolated and characterized. Several sequence polymorphisms have also been identified. FISH-mapping localized the HSP90AA1 gene on ovine chromosome OAR19q24dist, which was confirmed by linkage analysis. This chromosome region has been shown to include a quantitative trait loci (QTL) for scrapie incubation period in sheep. Expression analyses were carried out in spleen and cerebellum samples. No differences in the expression of the HSP90AA1 gene were found in any of these tissues (p > 0.05) between control and infected animal samples. Nevertheless, association analyses revealed that several polymorphisms in the 5' and 3' regions of the HSP90AA1 gene were differentially distributed among animals with different responses to scrapie infection. Thus, results presented here support the hypothesis that HSP90AA1 could be a positional and functional candidate gene modulating the response to scrapie in sheep.

Pathogenesis of bovine spongiform encephalopathy in sheep

The pathogenesis of bovine spongiform encephalopathy (BSE) in sheep was studied by immunohistochemical detection of scrapie-associated prion protein (PrPSc) in the gastrointestinal, lymphoid and neural tissues following oral inoculation with BSE brain homogenate. First accumulation of PrPSc was detected after 6 months in the tonsil and the ileal Peyer’s patches. At 9 months postinfection, PrPSc accumulation involved all gut-associated lymphoid tissues and lymph nodes as well as the spleen. At this time point, PrPSc accumulation in the peripheral neural tissues was first seen in the enteric nervous system of the caudal jejunum and ileum and in the coeliac-mesenteric ganglion. In the central nervous system, PrPSc was first detected in the dorsal motor nucleus of the nervus Vagus in the medulla oblongata and in the intermediolateral column in the spinal cord segments T7–L1. At subsequent time points, PrPSc was seen to spread within the lymphoid system to also involve all non-gut-associated lymphoid tissues. In the enteric nervous system, further spread of PrPSc involved the neural plexi along the entire gastrointestinal tract and in the CNS the complete neuraxis. These findings indicate a spread of the BSE agent in sheep from the enteric nervous system through parasympathetic and sympathetic nerves to the medulla oblongata and the spinal cord.

Cholesterol Metabolism in Brain and Skin Fibroblasts from Sarda Breed Sheep With Scrapie-resistant and Scrapie-susceptible Genotypes

Scrapie is a fatal spongiform encephalopathy of sheep, a transmissible form of prion disease caused by neuronal accumulation of the aberrantly conformed prion protein (PrPsc). Currently, no ante-mortem diagnostic tests are available to detect this untreatable disease in the pre-clinical stage, thus making difficult to control its spread. Recent evidence suggests that the production of PrPsc can be modulated by the levels of membrane cholesterol in neuronal cells. Since cholesterol levels in cell membranes are dependent on cholesterol homeostasis in the whole organism, we studied cholesterol metabolism in brain tissues, plasma and skin fibroblasts of Sarda breed sheep with scrapie-resistant (ARR/ARR) and scrapie-susceptible (ARQ/ARQ) prion protein genotypes, both not infected (ARQ/ARQ-) and infected (ARQ/ARQ+) with scrapie. We found that, the levels of cytoplasmic cholesterol esters (CE) in brains and skin fibroblasts from sheep with the ARQ/ARQ genotype were consistently higher than those from sheep with the ARR/ARR genotype. Conversely, the levels of free cholesterol (FC) were lower in ARQ/ARQ, as compared to ARR/ARR sheep, thus resulting in a sharp reduction of the FC/CE ratio. Moreover, both uninfected and infected ARQ/ARQ sheep showed abnormally low levels of high density lipoprotein-cholesterol (HDL-C) in their plasma, as compared to ARR/ARR sheep. These data other than adding new strength to the notion that altered levels of intracellular cholesterol may indicate the presence of a lipid metabolic state that predisposes to infection with, and accumulation of, PrPsc in the brain, discriminate for the first time between two distinct but related cellular pools of cholesterol, namely membrane FC on one hand and cytoplasmic CE on the other.

Genotyping of Ovine Prion Protein Gene (PRNP) Variants by PCR with Melting Curve Analysis,

Scrapie is the transmissible spongiform encephalopathy in sheep. Because genetic variants of the ovine PrP gene (PRNP) can be associated with disease risk, the European Union initiated programs to eradicate high-risk PRNP genotypes from sheep livestock. For this purpose, reliable and cost-effective genotyping is needed. We amplified DNA to cover the 3 risk codons in exon 3 encoding amino acids 136, 154, and 171. Amplicons were mixed with dye-labeled probe sets, and melting curves were recorded in a LightCycler by use of color and temperature multiplexing. Probe design was based on thermodynamic calculations to ensure unequivocal results for the 3 codons of interest, taking the additional F141 and T137 sequence variants into account. The fluorescence resonance energy transfer (FRET) method, when compared with sequencing, gave exactly the predicted melting temperatures for all possible genotypes. When we validated the method with samples from official certification programs, it showed completely matching results. Turnaround time was approximately 5 h after receipt of a whole-blood sample. The method detected the rare sequence variants T137 and F141, which were clearly distinguishable from the other known genotypes by melting curve analysis. One scrapie sheep was ARR/ARR, which is considered the haplotype with the lowest risk. The FRET-based PRNP genotyping method for sheep is rapid and can differentiate all genotypes at each locus in 1 capillary. The assay is fast and has lower costs than restriction fragment length polymorphism analysis or sequencing.

Monitoring for bovine spongiform encephalopathy in sheep in Great Britain, 1998–2004

Bovine spongiform encephalopathy (BSE) may have been transmitted to British sheep via contaminated feed in the 1980s. Strain-typing techniques based on immunohistochemical (IHC) detection of abnormal protein (PrP(d)) and the molecular analysis of proteinase-resistant protein (PrP(res)) by Western blotting (WB) can discriminate between natural or experimental scrapie and experimental BSE in sheep. Between 1 January 1998 and 31 October 2001, 1247 sheep, clinically suspected of scrapie, were found to be positive by statutory tests in Great Britain. Archived brain tissue from these cases was retested by using these discriminatory methods. Twelve brain samples showed PrP(res) WB patterns that were unlike those found in natural or experimental scrapie. Prospective screening of fresh tissue from a further 1121 scrapie cases was also carried out between 1 November 2001 and 31 May 2004. Two samples gave WB results with similarities to the results found for experimental BSE in sheep. When all 14 unusual cases were tested by IHC, no match to experimental BSE in sheep was found. There were uncertainties within the retrospective study, where some equivocal results were obtained due to poor tissue quality or the unavailability of the optimum brain region. However, for the samples where tissue condition was optimum, our results provide no evidence for the presence of BSE in sheep. Epidemiological interpretation of the 450 flocks sampled indicates that the maximum proportion of sheep transmissible spongiform encephalopathy cases that could be BSE is 0.66%. This estimate is lower than calculated previously (5%), when the analysis was based on the results of strain typing in mice.

Estimates of the prevalence of transmissible spongiform encephalopathies in sheep and goats in France in 2002

An active surveillance programme for transmissible spongiform encephalopathies (TSEs) in sheep and goats was implemented in France in 2002 at abattoirs and rendering plants. The analysis of the results of...

Recognition of the Nor98 Variant of Scrapie in the Swedish Sheep Population

Within the framework of the active surveillance for transmissible spongiform encephalopathies in sheep in Sweden, 4 cases of the atypical form of scrapie, Nor98, were identified during 2003. Nor98 is a recently recognized and poorly understood variant of scrapie, first described in Norway. The cases were positive by the rapid test (enzyme-linked immunosorbent assay). Immunohistochemical staining showed diffuse thin-granular staining of the cerebellar cortex. Western immunoblotting analysis of specimens of brain stem and cerebellum showed a light band of approximately 12 kDa. Typical scrapie was ruled out based on the confirmatory testing. The affected ewes were from 4 different flocks. They were between 7 and 9 years old. Two were of the ARQ/ARQ genotype, 1 ARR/ARQ, and 1 ARR/AHQ. Two ewes had shown ataxia, and the other 2 had no clinical signs. Whole-flock slaughter was applied, and testing of the flock mates did not reveal additional cases. Nor98 differs from typical scrapie in its epidemiology, frequency of genotypes of sheep affected, clinical signs, microscopic lesions, distribution of scrapie prion protein in the brain, and characteristics of the immunostaining and immunoblotting profiles.

Discrimination between scrapie and bovine spongiform encephalopathy in sheep by molecular size, immunoreactivity, and glycoprofile of prion protein.

A procedure for discrimination between scrapie and bovine spongiform encephalopathy (BSE) in sheep is of importance for establishing whether BSE has entered the sheep population. Since BSE has not yet been found in sheep at the farm level, such discrimination procedures can be developed only with experimental sheep BSE. Two distinctive molecular features of the prion protein (PrP)-molecular size and glycosylation profile-in proteinase K digests of brain stem tissue from sheep were used here; upon Western blotting, these features led to an unequivocal discrimination among natural scrapie, experimental scrapie, and experimental BSE. The higher electrophoretic mobility of PrP in sheep BSE could be best observed after deglycosylation treatment with N-glycosidase F. A simpler method for confirmation of this size difference involved comparison of the ratios for the binding of two monoclonal antibodies: P4 and 66.94b4. Based on epitope mapping studies with P4 and peptides, it appeared that N-terminal amino acid sequence WGQGGSH was intact only in sheep scrapie digests. Another feature typical for PrP in sheep BSE was the large fraction of diglycosylated PrP (70% or more). These data were obtained for a large group of positive sheep, consisting of 7 sheep with experimental BSE infection (genotypes: six ARQ/ARQ and one AHQ/AHQ), 48 sheep naturally infected with scrapie (six different genotypes), and 3 sheep with primary experimental scrapie infection. Routine tests of slaughter material serve well for the initial detection of both BSE and scrapie. With Western blotting as a rapid follow-up test, a 66.94b4/P4 antibody binding ratio above 1.5 is a practical indicator for serious suspicion of BSE infection in sheep.