Genotyping and surveillance for scrapie in Finnish sheep.
ABSTRACT: BACKGROUND: The progression of scrapie is known to be influenced by the amino acid polymorphisms of the host prion protein (PrP) gene. There is no breeding programme for TSE resistance in sheep in Finland, but a scrapie control programme has been in place since 1995. In this study we have analysed PrP genotypes of total of 928 purebred and crossbred sheep together with the data of scrapie survey carried out in Finland during 2002-2008 in order to gain knowledge of the genotype distribution and scrapie prevalence in Finnish sheep. RESULTS: The ARQ/ARQ genotype was the most common genotype in all breeds studied. ARR allele frequency was less than 12% in purebred Finnish sheep and in most genotypes heterozygous for ARR, the second allele was ARQ. The VRQ allele was not detected in the Grey race sheep of Kainuu or in the Aland sheep, and it was present in less than 6% of the Finnish Landrace sheep. Leucine was the most prominent amino acid found in codon 141. In addition, one novel prion dimorphisms of Q220L was detected. During the scrapie survey of over 15 000 sheep in 2002-2008, no classical scrapie cases and only five atypical scrapie cases were detected. CONCLUSIONS: The results indicate that the Finnish sheep populations have genetically little resistance to classical scrapie, but no classical scrapie was detected during an extensive survey in 2002-2008. However, five atypical scrapie cases emerged; thus, the disease is present in the Finnish sheep population at a low level.
Project description:The susceptibility of sheep to scrapie is under the control of the host's prion protein (PrP gene and is also influenced by the strain of the agent. PrP polymorphisms at codons 136 (A/V), 15 (R/H) and 171 (Q/R/H) are the main determinants of susceptibility/resistance of sheep to classical scrapie. They are combined in four main variants of the wild-type ARQ allele: VRQ, AHQ, ARH and ARR. Breeding programmes have been undertaken on this basis in the European Union and th USA to increase the frequency of the resistant ARR allele in sheep populations. Herein, we report th results of a multi-flock study showing the protective effect of polymorphisms other than those a codons 136, 154 and 171 in Sarda breed sheep. All ARQ/ARQ affected sheep (n = 154) and 37 negative ARQ/ARQ controls from four scrapie outbreaks were submitted to sequencing of the Pr gene. The distribution of variations other than those at the standard three codons, between scrapie cases and negative controls, was statistically different in all flocks. In particular, the AT(137)RQ an ARQK(176) alleles showed a clear protective effect. This is the first study demonstrating a protective influence of alleles other than ARR under field conditions. If further investigations in other sheep breeds and with other scrapie sources confirm these findings, the availability of various protective alleles in breeding programmes of sheep for scrapie resistance could be useful in breeds with a low frequency of the ARR allele and would allow maintaining a wider variability of the PrP gene.
Project description:BACKGROUND: In sheep, susceptibility to scrapie, which is similar to human prion diseases such as Kuru and variant Creutzfeldt-Jakob disease (vCJD), is determined by prion protein (PrP) gene (Prnp) polymorphisms. Sheep with genotype ARQ/ARQ, denoting polymorphisms at codons 136, 154, and 171, are susceptible, whereas those with genotypes ARR/ARQ and ARR/ARR are resistant, indicating dominance of ARR over the ARQ allele. AIMS: Based on familial CJD E200K, 129V, where preferential use of the 200E allele in EK heterozygous individuals confers resistance, heterozygous ARR/ARQ sheep were used to test the hypothesis that resistance is caused by preferential use of the ARR allele. METHODS: After assessment of equivalent PrP expression across genotypes, allele use was analysed by sequencing reverse transcription polymerase chain reaction derived DNA clones containing the Prnp gene coding sequence. RESULTS: The ARR to ARQ ratio was 1.1 in 133 clones, representing Prnp mRNA from three ARR/ARQ sheep, indicating equal use of both alleles. CONCLUSIONS: Dominance of the resistant associated allele in sheep scrapie involves mechanisms other than the absence of PrP derived from the disease associated ARQ allele.
Project description:The susceptibility of sheep to classical scrapie and bovine spongiform encephalopathy (BSE) is mainly influenced by prion protein (PrP) polymorphisms A136V, R154H, and Q171R, with the ARR allele associated with significantly decreased susceptibility. Here we report the protective effect of the amino acid substitution M137T, I142K, or N176K on the ARQ allele in sheep experimentally challenged with either scrapie or BSE. Such observations suggest the existence of additional PrP alleles that significantly decrease the susceptibility of sheep to transmissible spongiform encephalopathies, which may have important implications for disease eradication strategies.
Project description:Sheep with valine (V) at codon 136 and glutamine (Q) at codon 171 of the prion protein gene ( Prnp) are highly susceptible to classical scrapie, whereas phenylalanine (F) at codon 141 and histidine (H) at codon 154 play a major role in the susceptibility to atypical scrapie. A TaqMan real-time PCR assay was developed to determine Prnp alleles at codons 136, 141, 154, and 171 and used in classical scrapie eradication and breeding programs adopted in Slovenia. The frequency of the most resistant genotypes ARR/ARR and ARR/ARQ increased significantly in tested animals ( n = 35,138) from 6.7 and 27.1% of the tested sheep in 2006 to 12.1 and 32.4%, respectively, in 2015. Frequencies of more susceptible genotypes ARQ/ARQ and ARQ/VRQ decreased significantly from 36.4 and 3.5% in 2006 to 31.1 and 1.8%, respectively, in 2015. The most susceptible genotype VRQ/VRQ was detected in <0.5% of tested sheep. Frequencies of alleles AFRQ and AHQ affecting the susceptibility to atypical scrapie did not change significantly. The developed assay was suitable for genotyping on a small-to-medium throughput scale and was successfully used in classical scrapie eradication, as well as for the selection of classical scrapie-resistant sheep within breeding programs in Slovenia.
Project description:<h4>Background</h4>Prion protein (PrP) alleles associated with scrapie susceptibility persist in many sheep populations even with high frequencies despite centuries of selection against them. This suggests that scrapie susceptibility alleles have a pleiotropic effect or are associated with fitness or other traits that have been subject to selection.<h4>Methodology/principal findings</h4>We genotyped all lambs in two scrapie-free Scottish Blackface sheep flocks for polymorphisms at codons 136, 154 and 171 of the PrP gene. We tested potential associations of the PrP genotype with lamb viability at birth and postnatal survival using a complementary log-log link function and a Weibull proportional hazard model, respectively. Here we show there is an association between PrP genotype, as defined by polymorphisms at codons 154 ad 171, and postnatal lamb survival in the absence of scrapie. Sheep carrying the wild-type ARQ allele have higher postnatal survival rates than sheep carrying the more scrapie-resistant alleles (ARR or AHQ).<h4>Conclusion</h4>The PrP genotypes associated with higher susceptibility to scrapie are associated with improved postnatal survival in the absence of the disease. This association helps to explain the existence, and in many instances the high frequency, of the ARQ allele in sheep populations.
Project description:Ovine PBMCs (peripheral blood mononuclear cells) express PrP(C) [cellular PrP (prion-related protein)] and have the potential to harbour and release disease-associated forms of PrP during scrapie in sheep. Cell-surface PrP(C) expression by PBMCs, together with plasma PrP(C) levels, may contribute to the regulatory mechanisms that determine susceptibility and resistance to natural scrapie in sheep. Here, we have correlated cell-surface PrP(C) expression on normal ovine PBMCs by FACS with the presence of PrP(C) in plasma measured by capture-detector immunoassay. FACS showed similar levels of cell-surface PrP(C) on homozygous ARR (Ala136-Arg154-Arg171), ARQ (Ala136-Arg154-Gln171) and VRQ (Val136-Arg154-Gln171) PBMCs. Cell-surface ovine PrP(C) showed modulation of N-terminal epitopes, which was more evident on homozygous ARR cells. Ovine plasma PrP(C) levels showed genotypic variation and the protein displayed C-terminal epitopes not available in cell-surface PrP(C). Homozygous VRQ sheep showed the highest plasma PrP(C) level and homozygous ARR animals the lowest. For comparison, similar analyses were performed on normal bovine PBMCs and plasma. PrP(C) levels in bovine plasma were approx. 4-fold higher than ovine homozygous ARQ plasma despite similar levels of PBMC cell-surface PrP(C) expression. Immunoassays using C-terminal-specific anti-PrP monoclonal antibodies as capture and detector reagents revealed the highest level of PrP(C) in both ovine and bovine plasma, whilst lower levels were detected using N-terminal-specific monoclonal antibody FH11 as the capture reagent. This suggested that a proportion of plasma PrP(C) was N-terminally truncated. Our results indicate that the increased susceptibility to natural scrapie displayed by homozygous VRQ sheep correlates with a higher level of plasma PrP(C).
Project description:Scrapie is a transmissible spongiform encephalopathy that occurs in sheep. Atypical/Nor98 scrapie occurs in sheep that tend to be resistant to classical scrapie and it is thought to occur spontaneously. The purpose of this study was to test the transmission of the Atypical/Nor98 scrapie agent in three genotypes of Suffolk sheep and characterize the distribution of misfolded prion protein (PrPSc). Ten sheep were intracranially inoculated with brain homogenate from a sheep with Atypical/Nor98 scrapie. All sheep with the ARQ/ARQ and ARQ/ARR genotypes developed Atypical/Nor98 scrapie confirmed by immunohistochemistry, and one sheep with the VRQ/ARQ genotype had detectable PrPSc consistent with Atypical/Nor98 scrapie at the experimental endpoint of 8 years. Sheep with mild early accumulations of PrPSc in the cerebellum had concomitant retinal PrPSc. Accordingly, large amounts of retinal PrPSc were identified in clinically affected sheep and sheep with dense accumulations of PrPSc in the cerebellum.
Project description:This report describes the genetics of the prion protein gene (PRNP) at codons 136, 154, and 171 for sheep diagnosed with naturally acquired classical scrapie in Canada between 1998 and 2008. Genotyping analysis was performed on 249 sheep with confirmed classical scrapie infection representing 98 flocks from 6 provinces. A further case-control analysis of 3 of these flocks compared the genotypes between infected sheep (n = 72) and those of their healthy flockmates (n = 1990). The incidence of classical scrapie in the Canadian sheep population was highly associated with the ARQ haplotype (91.8%) and the ARQ/ARQ genotype (91.6%). In addition, the ARQ haplotype was found at significantly higher frequency in scrapie-infected sheep when compared with their healthy flockmates. Comparison with other published data suggests that the scrapie risk of PRNP genotypes differs between Canada and countries where the VRQ allele is associated with the highest susceptibility to infection.
Project description:BACKGROUND: Diagnosis based on prion detection in lymph nodes of sheep and goats can improve active surveillance for scrapie and, if it were circulating, for bovine spongiform encephalopathy (BSE). With sizes that allow repetitive testing and a location that is easily accessible at slaughter, retropharyngeal lymph nodes (RLN) are considered suitable organs for testing. Western blotting (WB) of brain homogenates is, in principle, a technique well suited to both detect and discriminate between scrapie and BSE. In this report, WB is developed for rapid diagnosis in RLN and to study biochemical characteristics of PrPres. RESULTS: Optimal PrPres detection in RLN by WB was achieved by proper tissue processing, antibody choice and inclusion of a step for PrPresconcentration. The analyses were performed on three different sheep sources. Firstly, in a study with preclinical scrapie cases, WB of RLN from infected sheep of VRQ/VRQ genotype--VRQ represents, respectively, polymorphic PrP amino acids 136, 154, and 171--allowed a diagnosis 14 mo earlier compared to WB of brain stem. Secondly, samples collected from sheep with confirmed scrapie in the course of passive and active surveillance programmes in the period 2002-2003 yielded positive results depending on genotype: all sheep with genotypes ARH/VRQ, VRQ/VRQ, and ARQ/VRQ scored positive for PrPres, but ARQ/ARQ and ARR/VRQ were not all positive. Thirdly, in an experimental BSE study, detection of PrPres in all 11 ARQ/ARQ sheep, including 7 preclinical cases, was possible. In all instances, WB and IHC were almost as sensitive. Moreover, BSE infection could be discriminated from scrapie infection by faster electrophoretic migration of the PrPres bands. Using dual antibody staining with selected monoclonal antibodies like 12B2 and L42, these differences in migration could be employed for an unequivocal differentiation between BSE and scrapie. With respect to glycosylation of PrPres, BSE cases exhibited a greater diglycosylated fraction than scrapie cases. Furthermore, a slight time dependent increase of diglycosylated PrPres was noted between individual sheep, which was remarkable in that it occurred in both scrapie and BSE study. CONCLUSION: The present data indicate that, used in conjunction with testing in brain, WB of RLN can be a sensitive tool for improving surveillance of scrapie and BSE, allowing early detection of BSE and scrapie and thereby ensuring safer sheep and goat products.
Project description:Classical scrapie is a prion disease in sheep and goats. In sheep, susceptibility to disease is genetically influenced by single amino acid substitutions. Genetic breeding programs aimed at enrichment of arginine-171 (171R) prion protein (PrP), the so-called ARR allele, in the sheep population have been demonstrated to be effective in reducing the occurrence of classical scrapie in the field. Understanding the molecular basis for this reduced prevalence would serve the assessment of ARR adaptation. The prion formation mechanism and conversion of PrP from the normal form (PrP(C)) to the scrapie-associated form (PrP(Sc)) could play a key role in this process. Therefore, we investigated whether the ARR allele substantially contributes to scrapie prion formation in naturally infected heterozygous 171Q/R animals. Two methods were applied to brain tissue of 171Q/R heterozygous sheep with natural scrapie to determine the relative amount of the 171R PrP fraction in PrP(res), the proteinase K-resistant PrP(Sc) core. An antibody test differentiating between 171Q and 171R PrP fragments showed that PrP(res) was mostly composed of the 171Q allelotype. Furthermore, using a novel tool for prion research, endoproteinase Lys-C-digested PrP(res) yielded substantial amounts of a nonglycosylated and a monoglycosylated PrP fragment comprising codons 114 to 188. Following two-dimensional gel electrophoresis, only marginal amounts (<9%) of 171R PrP(res) were detected. Enhanced 171R(res) proteolytic susceptibility could be excluded. Thus, these data support a nearly zero contribution of 171R PrP in PrP(res) of 171R/Q field scrapie-infected animals. This is suggestive of a poor adaptation of classical scrapie to this resistance allele under these natural conditions.