The genetics of scrapie in sheep and goats

Scrapie, an invariably fatal disease of sheep and goats, is a transmissible spongiform encephalopathy (TSE). The putative infectious agent is the host-encoded prion protein, PrP. The development of scrapie is closely linked to polymorphisms in the host PrP gene. The pathogenesis of most TSEs involves conversion of normal, cellular PrP into a protease-resistant, pathogenic isoform called PrPSc. The conversion to PrPSc involves change in secondary structure; it is impacts on these structural changes that may link polymorphisms to disease. Within the structured C-terminal part of PrP polymorphisms have been reported at 15 and 10 codons of the sheep and goat PrP genes respectively. Three polymorphisms in sheep are acutely linked to the occurrence of scrapie: A136V, R154H and Q171R/H. These generate five commonly observed alleles: ARQ, ARR, AHQ, ARH and VRQ. ARR and AHQ are associated with resistance; ARQ, ARH and VRQ are associated with susceptibility. There are subtle effects of specific allele pairings (genotypes). Generally, more susceptible genotypes have younger ages at death from scrapie. Different strains of scrapie occur which may attack genotypes differently. Different sheep breeds vary in the assortment of the five alleles that they predominantly encode. The reason for this variation is not known. Furthermore, certain genotypes may be susceptible to scrapie in some breeds and resistant in others. The explanation is not known, but may relate to different scrapie strains circulating in different breeds, or there may be effects of other genes which modulate the effect of PrP.     

Molecular cloning and polymorphism analysis of the prion protein gene in Tan sheep of Ningxia, China

The resistance or susceptibility of sheep to scrapie is associated with polymorphisms of the prion protein gene (PRNP), particularly, single nucleotide polymorphisms (SNPs) in amino acid positions 136, 154 and 171. The prion protein (PrP) gene sequence and the deduced amino acid alignment of prion protein in Tan sheep, a local Chinese sheep breed traditionally raised in Ningxia, northwestern China, were determined and variability of the PrP amino acids sequence was analyzed in this study. The PrP nucleic acids and amino acids sequences of 112 Tan sheep were highly homogenous, although polymorphism of the PrP gene was detected at several sites, particularly codons 106, 154, and 171. The analysis of both sequences revealed that the most predominant allele at codons 136, 154 and 171 in Tan sheep was ARQ, which was known to be associated with high susceptibility to scrapie in sheep. The result suggests that Tan sheep is potentially susceptible to scrapie. Our findings provide valuable information for future breeding projects to scrapie resistance in Tan sheep.     

Prion protein alleles showing a protective effect on the susceptibility of sheep to scrapie and bovine spongiform encephalopathy

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.     

Prion protein (PrP) gene polymorphism in Red Maasai and Black Head Persian sheep breeds in Tanzania: Consistent profile regardless of locations

The status of scrapie in Africa is largely unknown. The susceptibility to scrapie and its pathology is determined by amino acid polymorphisms at positions 136 (A/V), 154 (R/H) and 171 (Q/R/H) of the ovine PrP gene (PrP genotype) of the animals. Despite the widely studied PrP gene polymorphisms worldwide, limited data is available on PrP genotypes of sheep from the African continent. Previously, we have reported six PrP genotypes derived from four different alleles in Red Maasai and Black Herd Persian (BHP), the ARQ/ARQ genotype being more frequent in the Red Maasai than in the BHP sheep. The highly susceptible VRQ allele was not found in any of the sheep breeds; the ARR allele was absent in the Red Maasai or occurred at a low frequency in the BHP. The lack of the highly susceptible VRQ alleles among Tanzanian sheep examined, necessitated further examinations on genetic susceptibility in sheep of the same breeds, but originating from an entirely different part of Tanzania. Consistently, ARQ/ARQ genotype was observed in 88% of Red Maasai and 64% of BHP sheep, ARQ/AHQ genotype was present in 12% in Red Maasai and 36% in BHP. Neither the highly resistant (ARR/ARR), nor the highly susceptible (VRQ/VRQ) genotypes were found. The ARQ and AHQ were the only alleles observed. The ARQ allele constituted 94%, 82% and 67% in the Red Maasai, BHP and cross-bred sheep, respectively. The AHQ constituted 6%, 18% and 33%, respectively. Data reported here, provide additional information on genetic susceptibility of the Red Maasai, BHP and their crosses; they may be helpful in policy formulation for future prevention of scrapie.     

新疆地区绵羊品种朊蛋白基因136、154和171位点的多态性

绵羊痒病是一种渐进性和致死性中枢神经系统疾病,绵羊朊蛋白基因(Prion protein gene,PRNP)多态性与痒病的易感或抗性有关,其中PRNP136位(V/A)、154位(H/R)和171位(H/Q/R)的基因多态性与该病发生最相关。为评价新疆地区主要绵羊品种对痒病的易感性,文章对新疆地区10个绵羊品种(阿勒泰、巴士拜、巴音布鲁克、多浪、和田、策勒黑、中国美利奴、德国肉用美利奴、特克赛尔和萨福克羊)共746只个体PRNP基因的136位(V/A)、154位(H/R)和171位(H/Q/R)的遗传多态性进行分析,检测到了ARQ、ARR、ARH、ARK、VRQ、AHR、AHQ、AHH8种等位基因,其中ARQ和ARR等位基因存在于所有品种中,且ARQ在所有品种中的基因频率最高。ARH存在于除萨福克和德国肉用美利奴羊外的8个绵羊品种中。仅在新疆地方品种阿勒泰、巴音布鲁克、巴士拜和多浪羊中检测到ARK等位基因。而VRQ、AHR、AHQ和AHH4种等位基因只在中国美利奴羊上存在,且频率极低。在10个品种中共检测到了ARQ/ARQ、ARQ/ARK、ARR/ARR、ARH/ARH、ARQ/ARR、ARH/ARQ、ARH/ARR、ARK/ARK、ARH/ARK、ARQ/VRQ、ARQ/AHQ、ARQ/AHR和ARH/AHH13种基因型,其中中度易感的ARQ/ARQ基因型频率最高,而抗性最强的ARR/ARR基因型仅存在于巴音布鲁克、策勒黑、中国美利奴、特克赛尔和德国肉用美利奴羊,且频率较低。文章首次在中国美利奴羊上发现了易感性很强的VRQ/ARQ基因型。上述结果提示新疆的主要绵羊品种对痒病的抗性较弱。     

The PrP genotype of sheep of the improved Valachian breed

In a worldwide majority of sheep breeds an excessive susceptibility to scrapie associated with the PrP gene alleles coding for valine (V; at the 136 codon) and glutamine (Q; at the 171 codon) (e.g., VRQ/VRQ, VRQ/ARQ, or ARQ/ARQ) was demonstrated. Particularly the PrPVRQ allele is closely associated with the high-risk development of the disease; the PrPARQ allele can also fulfill this function but under certain limited conditions. Polymorphism in the PrP gene sequences (conclusively related to the increased susceptibility of sheep to scrapie) of improved Valachian sheep from two Slovak regions, Orava and Spis, was determined. Examination of 735 sheep showed that ARR/ARQ was the most frequent genotype (45.2%). High-risk genotypes were determined in 32.4% of sheep (ARQ/ARQ 19.3, ARR/VRQ 9.0, ARR/VRQ 3.5, VRQ/VRQ 0.3, ARR/VRR 0.3). Low-risk genotypes were found in 67.7% of sheep (ARR/ARQ 45.2, ARR/ARR 10.9, ARR/AHQ 5.7, ARQ/ARQ 4.9, AHQ/AHQ 0.7, ARR/AHR 0.3). Despite the geographically distant flocks of improved Valachian sheep investigated no difference in the occurrence of individual PrP genotypes was observed.     

Genotyping of the prion protein (PrP) gene in Red Maasai and Black Head Persian sheep in Tanzania

Susceptibility to scrapie in sheep is largely influenced by four polymorphic amino acid positions of the ovine PrP gene at codons 136, 141, 154 and 171. Genotyping of corresponding DNA polymorphism can be used as a basis for selection decisions. A total of 100 Red Maasai and 79 Black Herd Persian sheep, representative of the widely distributed breeds in Tanzania, were genotyped by real-time PCR. Genomic DNA was extracted from buccal epithelial cells. We report six genotypes derived from four different alleles, with the ARQ/ARQ genotype being more frequent (p = 0.0081) in the Red Maasai than in the Black Head Persian sheep. Our study also demonstrated higher allelic frequency (p = 0.00055) of the ARQ in the Red Maasai than in Black Head Persian sheep, while the AHQ allelic frequency was higher (p = 0.00086) in the Black Head Persian. All the animals were homozygous LL₁₄₁. The highly susceptible VRQ allele was not found in any of the sheep breeds examined in this study. Both breeds were genetically low-level resistant to scrapie (NSP3). Due to absence or very low frequency of the ARR allele in the two breeds, selection for genetic resistance to scrapie through an increase of the ARR allele does not seem very relevant. If new breeds of sheep are to be introduced for crossbreeding in Tanzania, care should be taken to avoid import of the VRQ allele. To our knowledge, this is the first study exploring the PrP genotypes in sub-Saharan African sheep and in the Red Maasai and Black Head Persian sheep breeds in particular. The data provide base-line information on genetic susceptibility of the two sheep breeds to scrapie, which may be useful in policy formulation for prevention and future research on prion diseases.     

Screening for polymorphism at the prion protein (PrP) locus (PRNP) in Awassi and Assaf populations in Israel, the Palestinian Authority and Jordan

Screening for polymorphism at the prion protein (PrP) locus (PRNP) was carried out in 33 flocks of Local Awassi (LA) sheep in Israel, the Palestinian Authority (PA) and Jordan. In addition, PRNP genotyping was carried out in two flocks of Improved Awassi (IA) in Israel and the PA and in 11 flocks of Assaf sheep in Israel. Most of the rams present in the flocks at the time of the survey 328, 44 and 298 rams from the LA, IA and Assaf breeds, respectively, were genotyped. The ARQ allele was found to be predominant in all three breeds with average frequencies of 0.74, 0.78 and 0.86 for the LA, IA and Assaf breeds, respectively. Frequencies of the desirable ARR allele in these breeds were 0.10, 0.05 and 0.06, respectively. ARH, AHQ and ARK alleles were identified at low/absent frequencies. Only one Assaf ewe carried the undesirable V allele at codon 136. A few scrapie outbreaks have been documented in the past in the region. To increase ARR allele frequencies in the Awassi and Assaf populations, distribution of homozygous ARR/ARR rams is recommended.     

Frequencies of PrP genotypes and their implication for breeding against scrapie susceptibility in nine Pakistani sheep breeds

Prion protein (PrP) gene of 308 sheep was genotyped to investigate polymorphisms at scrapie-associated codons 136, 154 and 171 to assess the resistance of nine different Pakistani sheep breeds to natural/typical scrapie. As a result six genotypes were established on the basis of polymorphic codons 154 and 171. The most scrapie-susceptible codon 136 (A/V) was monomorphic (A) in all breeds. Wild-type genotype ARQ/ARQ was detected with maximum prevalence ranging from 63.2% in crossbred Pak-karakul to 100% in native Buchi, Kachi and Thalli breeds. The most frequent of typical scrapie-associated genotypes was ARQ/ARR as indicated by five of nine breeds. The coding region of PrP gene of 49 animals from the total sampled was also sequenced to ascertain additional polymorphisms. Polymorphism was found in 13 animals of the six breeds in codons 101(Q/R), 112(M/T), 146(N/S) and 189(Q/L) and ten genotypes were established on the basis of these polymorphic codons. Only Hissardale possessed five of the ten genotypes. The most frequent genotype was M¹¹²ARQ/T¹¹²ARQ detected in Hissardale, Pak-karakul and Awassi, whereas genotypes ARQr²³¹/ARQr²³¹ and ARQR²³¹/ARQr²³¹ (established on the basis of silent polymorphism agg/cgg-R/R) were detected in all breeds. Some animals consisted of three polymorphisms at different PrP codons that are not common in European breeds. An infrequent double heterozygosity (c/c a/g g/t) for codon 171 resulting in a genotype R/H was also detected in three animals each one from Kajli, Hissardale and Pak-karakul. This study concludes that all native sheep breeds are poor in scrapie-resistant PrP genotypes and could contract scrapie if exposed to prions.     

Genetic diversity of the prion protein gene (<Emphasis Type="Italic">PRNP</Emphasis>) coding sequence in Czech sheep and evaluation of the national breeding programme for resistance to scrapie in the Czech Republic

Of 34 breeds kept in the Czech Republic 45,604 sheep were genotyped for codons 136, 154 and 171 in the prion protein gene (PRNP) during the years 2006–2014. In this cohort, haplotypes ARR, ARQ, ARH, AHQ, VRQ, AHR and ARK were detected. The haplotype AF₁₄₁RQ associated with susceptibility to atypical scrapie was observed in nine out of 30 breeds analysed for this purpose. In addition, six rare nonsynonymous substitutions producing haplotypes AT₁₃₇RQ, AN₁₃₈RQ, AG₁₅₁RQ, AH₁₅₁RQ, ARL₁₆₈Q and ARQE₁₇₅ were identified in various breeds. Due to their low frequencies, these polymorphisms are of no potential importance for the breeding programme. With regard to their genetic particularity, Sumavka, Valachian and Cameroon breeds were screened for additional polymorphisms. Further haplotypes, AR₁₄₃RQ and AS₁₄₆RQ, were found in Sumavka and Cameroon, and in Valachian sheep, respectively. Frequencies of the ARR (resistance-associated), VRQ (susceptibility-associated) haplotypes, and of the most resistant ARR/ARR genotype calculated for sheep born in the years 2001–2003 and 2011–2013 documented effects of the 10 year-lasting national breeding programme. The total frequency of ARR doubled from 36.8 to 75.8 %, while the frequency of VRQ decreased from 4 to 0.7 %. The total frequency of the ARR/ARR genotype increased from 17.7 to 59 %. These data show that the national scrapie resistance breeding programme has had an important desirable effect on haplotype and genotype frequencies of PRNP in Czech sheep.     

Prion Protein Polymorphisms and Estimation of Risk of Scrapie in East Asian Sheep

Allele and genotype frequency distributions of prion protein (PrP) polymorphisms at three codons, 136, 154, and 171, in East Asian sheep were determined by PCR–RFLP analysis using 553 animals from nine local breeds of the northern group and four local breeds of the southern group. Based on the genotype distribution, the risk score for scrapie was estimated. Among the local breeds, ARQ appeared predominantly (0.7701–1), followed by ARH and ARR. From such a biased allele distribution, it was difficult to ascertain the prevalent genetic relationships. A marked difference in allele frequencies between the northern and southern groups was seen (P < 0.0001). The East Asian sheep had ARQ at the highest frequency (0.8834); in European sheep it was 0.5317. According to an assessment of scrapie risk in the PrP genotype classes, the predominant ARQ/ARQ in East Asian sheep corresponded to the risk score of R4. This finding suggests that East Asian sheep have a high level of genetic susceptibility to scrapie.     

Linkage disequilibrium across six prion gene regions spanning 20 kbp in U.S. sheep

Single nucleotide polymorphisms (SNPs) and haplotype alleles within the prion gene (PRNP) coding sequence of domestic sheep (Ovis aries) are associated with genetic predisposition to scrapie, a transmissible spongiform encephalopathy disease of sheep. This report describes regions of linkage disequilibrium (LD) throughout the PRNP gene region in U.S. sheep and provides a genetic framework for identifying additional PRNP determinants associated with scrapie resistance. Four sequence tagged sites (i.e., STS or amplicons) totaling 3869 bp and spanning 20 kbp of genomic PRNP sequence were sequenced in a diverse panel of 90 sires representing ten popular U.S. breeds of sheep. Analysis of these sequences identified 36 previously unreported polymorphisms. In combination with two previously characterized STS, 62 polymorphisms were analyzed in a 20-kbp PRNP region in this panel of U.S. sheep. Two regions of strong LD and ten common haplotypes were identified. The haplotype encoding amino acid residues A, R, and Q at codons 136, 154, and 171, respectively, was observed on nine larger haplotypes spanning PRNP from the promoter region to the 3′ untranslated region. The haplotype encoding VRQ was observed on two larger haplotypes, whereas ARR, ARH, and AHQ were each present on a single haplotype. The existence of multiple haplotypes encoding ARQ raises the question of whether sheep bearing these different haplotypes are equally susceptible to scrapie. The haplotype structure within the 20-kbp region of PRNP identified in this study is important for higher-resolution analysis of genetics contributions to scrapie susceptibility. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number DQ077504.     

Association between PrP genotypes and performance traits in a Welsh Mountain flock

The UK national scrapie plan (NSP) for sheep is based on selection for the resistant ARR/ARR genotype and elimination of susceptible types of the ovine prion protein (PrP) gene. The aim of this study was to estimate the possible association of the PrP genotype and performance traits by using data from the CAMDA Welsh Mountain flock. Four alleles (ARH, ARQ, ARR and VRQ) and 10 genotypes covering all five NSP risk groups were present in the CAMDA flock. Overall, the most common allele was ARR (35.2%), and VRQ was the least common (5.4%). The commonest genotypes were ARR/ARQ (23.7%) and ARR/AHQ (23.1%). The most resistant genotype, ARR/ARR, and the most susceptible genotype, VRQ/VRQ, were found in 10.2% and 0.3%, respectively, of the population tested. The associations of PrP genotypes with weight and ultrasonically scanned traits were investigated in three analyses, the first using genotypes, the second using risk categories and the third using number of alleles. These associations were evaluated by univariate analysis of each trait using an animal model with maternal effects where appropriate, and PrP was included as a fixed effect. Selection for scrapie resistance will not adversely affect progress in the traits considered and is consistent with improvements in muscle depth.     

Assessment of the genetic susceptibility of sheep to scrapie by protein misfolding cyclic amplification and comparison with experimental scrapie transmission studies

The susceptibility of sheep to scrapie is influenced mainly by the prion protein polymorphisms A136V, R154H, and Q171R/H. Here we analyzed the ability of protein misfolding cyclic amplification (PMCA) to model the genetic susceptibility of sheep to scrapie. For this purpose, we studied the efficiency of brain homogenates from sheep with different PrP genotypes to support PrP^Sc amplification by PMCA using an ARQ/ARQ scrapie inoculum. The results were then compared with those obtained in vivo using the same sheep breed, genotypes, and scrapie inoculum. Genotypes associated with susceptibility (ARQ/ARQ, ARQ/AHQ, and AHQ/ARH) were able to sustain PrP^Sc amplification in PMCA reactions, while genotypes associated with resistance to scrapie (ARQ/ARR and ARR/ARR) were unable to support the in vitro conversion. The incubation times of the experimental infection were then compared with the in vitro amplification factors. Linear regression analysis showed that the efficiency of in vitro PrP^Sc amplification of the different genotypes was indeed inversely proportional to their incubation times. Finally, the rare ARQK₁₇₆/ARQK₁₇₆ genotype, for which no in vivo data are available, was studied by PMCA. No amplification was obtained, suggesting ARQK₁₇₆/ARQK₁₇₆ as an additional genotype associated with resistance, at least to the isolate tested. Our results indicate a direct correlation between the ability of different PrP genotypes to undergo PrP^C-to-PrP^Sc conversion by PMCA and their in vivo susceptibility and point to PMCA as an alternative to transmission studies and a potential tool to test the susceptibility of numerous sheep PrP genotypes to a variety of prion sources.     

Frequencies of PrP gene haplotypes in British sheep flocks and the implications for breeding programmes

AIMS: To analyse the frequencies of prion (PrP) gene haplotypes in UK sheep flocks and evaluate their relevance to transmissible spongiform encephalopathies (TSEs) and TSE resistance breeding programmes in sheep. METHODS AND RESULTS: Genomic DNA isolated from sheep blood was PCR amplified for the coding region of the PrP gene and then sequenced. This study has analysed the sequence of PrP between codons 110 and 245 in 6287 ARQ haplotypes revealing a total of eight variant sequences, which represent a higher than expected 41% of all ARQ haplotypes. The additional PrP gene dimorphisms were M112T, L141F, M137T, H143R, H151C, P168L, Q175E and P241S. CONCLUSION: The results do not suggest a correlation between the occurrence of a specific ARQ haplotype and the scrapie disease status of a flock. The ARQ haplotype variability appears to be different in the UK sheep flocks compared with sheep flocks from outside the UK. SIGNIFICANCE AND IMPACT OF THE STUDY: Additional PrP dimorphisms may impact on the methodologies used for standard PrP genotyping in sheep breeding programmes. Some of these polymorphisms were found with significant frequencies in the UK sheep flocks and should therefore be considered in breeding programmes.     

Review: A review on classical and atypical scrapie in caprine: Prion protein gene polymorphisms and their role in the disease

Scrapie is a naturally occurring transmissible spongiform encephalopathy in sheep and goat. It has been known for ~250 years and is characterised by the accumulation of an abnormal isoform of a host-encoded prion protein that leads to progressive neurodegeneration and death. Scrapie is recognised in two forms, classical and atypical scrapie. The susceptibility to both types of scrapie is influenced by polymorphisms of the prion protein gene (PRNP). Sheep susceptibility or resistance to classical scrapie is strongly regulated by the polymorphisms at codons 136, 154 and 171 of the PRNP. The genetic role in atypical scrapie in sheep has been defined by polymorphisms at codons 141, 154 and 171, which are associated with different degrees of risk in the occurrence of the ovine disease. Progress has been achieved in the prevention of scrapie in sheep due to efficient genetic breeding programmes based on eradication and control of the disease. In Europe, the success of these programmes has been verified by applying eradication and genetic selection plans. In general terms, the ovine selection plans aim to eliminate and reduce the susceptible allele and to enrich the resistant allele ARR. During outbreaks all susceptible animals are slaughtered, only ARR/ARR resistant rams and sheep and semi-resistant females are preserved. In the occurrence of scrapie positive goats a complete cull of the flock (stamping out) is performed with great economic loss and severe risk of extinction for the endangered breeds. The ability to select scrapie-resistant animals allows to define new breeding strategies aimed to boost genetic progress while reducing costs during scrapie outbreaks. Allelic variants of PRNP can be protective for caprine scrapie, and the knowledge of their distribution in goats has become very important. Over the past few years, the integration of genetic information on goat populations could be used to make selection decisions, commonly referred to as genetic selection. The objective of this review was to summarise the main findings of polymorphisms of the caprine prion protein (PrP) gene and to discuss the possible application of goat breeding schemes integrating genetic selection, with their relative advantages and limitations.     

Characterization of the PRNP gene locus in Chios dairy sheep and its association with milk production and reproduction traits

The objective of this study was to examine the prion protein gene locus (PRNP) in Chios sheep. PRNP is linked with scrapie resistance in small ruminants. Here, its impact on milk production (test-day and total lactation yield) and reproduction (age at first lambing, conception rate at first service, and prolificacy) was assessed. Genotyping at codons 136, 154 and 171 (classical scrapie) and 141 (atypical scrapie) was performed using DNA from milk somatic cells and PCR-RFLP analysis. A total of 1013 Chios ewes raised in 23 flocks were used. This constituted a random sample of the national breeding population. A total of 15 genotypes and 6 alleles linked to codons 136, 154 and 171 were detected. All animals were homozygous for the leucine allele at codon 141. Linear mixed models were used to assess the impact of PRNP genotypes and alleles on milk production and reproduction traits. The TRQ allele, whose association with such traits was assessed for the first time, had an adverse effect on age at first lambing. All other PRNP alleles, including ARR, which is associated with increased resistance to classical scrapie, had no significant effect on the traits studied. No significant associations of the PRNP genotypes with production and reproduction traits were observed. It was concluded that selection for scrapie-resistant sheep is not expected to affect the ongoing breeding programme that aims to enhance the milk yield and reproduction of the Chios breed.     

Allelic variants of ovine prion protein gene (PRNP) in Oklahoma sheep

1,144 sheep belonging to 21 breeds and known crosses were sequence analyzed for polymorphisms in the ovine PRNP gene. Genotype and allele frequencies of polymorphisms in PRNP known to confer resistance to scrapie, a fatal neurodegenerative disease of sheep, are reported. Known polymorphisms at codons 136 (A/V), 154 (H/R) and 171 (Q/R/H/K) were identified. The frequency of the 171R allele known to confer resistance to type C scrapie was 53.8% and the frequency of the 136A allele known to influence the resistance to type A scrapie was 96.01%. In addition, we report the identification of five new polymorphisms at codons 143 (H/R), 167 (R/S), 180 (H/Y), 195 (T/S) and 196 (T/S). We also report the identification of a novel allele (S/R) at codon 138.     

Characterization of <Emphasis Type="Italic">PRNP</Emphasis> and <Emphasis Type="Italic">SPRN</Emphasis> coding regions from atypical scrapie cases diagnosed in Poland

Scrapie, a fatal transmissible spongiform encephalopathy (TSE) occurs in two phenotypes: classical and atypical. Many authors point out that the polymorphism of three codons (136, 154, 171) of the PRNP (PrP gene) is associated with a sheep susceptibility to classical scrapie. Until now, only one PRNP gene variant coding phenylalanine at codon 141 has been found to be associated with atypical scrapie. Another recently identified and interesting candidate gene for scrapie susceptibility in sheep is an SPRN gene coding for Shadoo protein (Sho). Sho is a highly interspecies conserved protein and an insertion/deletion (indel) found in a sheep Sho gene was associated with classical scrapie occurrence. Here we determined the polymorphism of PRNP and SPRN genes in nine atypical scrapie cases (six in native born sheep and three in imported sheep) and compared these results with a control group of healthy animals comprising six corresponding Polish sheep breeds. In atypical scrapie cases five PRNP diplotypes were identified: A₁₃₆R₁₅₄Q₁₇₁/ARQ, AHQ/ARQ, ARR/ARQ, ARR/AHQ and AHQ/AHQ. The ARR/AHQ diplotype was found only in imported sheep. A previously unobserved SNP in PRNP (E224K) was also found in both atypical scrapie and in a few control animals. In the ORF of the SPRN gene, six SNPs and one indel were identified. None of these variations was exclusive for scrapie animals and they were probably, naturally occurring polymorphisms. Special attention was given to the 6-bp indel SPRN polymorphism which was previously associated with classical scrapie occurrence.     

Comparative Susceptibility of Sheep of Different Origins,Breeds and PRNP Genotypes to Challenge with Bovine Spongiform Encephalopathy and Scrapie

Sheep are natural hosts of the prion disease, scrapie. They are also susceptible to experimental challenge with various scrapie strains and with bovine spongiform encephalopathy (BSE), which affects cattle and has been accidentally transmitted to a range of other species, including man. Incidence and incubation period of clinical disease in sheep following inoculation is controlled by the PRNP gene, which has different alleles defined on the basis of polymorphisms, particularly at codons 136, 154 and 171, although other codons are associated with survival time, and the exact responses of the sheep may be influenced by other breed-related differences. Here we report the results of a long term single study of experimental scrapie and BSE susceptibility of sheep of Cheviot, Poll Dorset and Suffolk breeds, originating from New Zealand and of a wide range of susceptible and resistant PRNP genotypes. Responses were compared with those of sheep from a closed Cheviot flock of UK origin (Roslin Cheviot flock). The unusually long observation period (6–8 years for most, but up to 12 years for others) allows us to draw robust conclusions about rates of survival of animals previously regarded as resistant to infection, particularly PRNP heterozygotes, and is the most comprehensive such study reported to date. BSE inoculation by an intracerebral route produced disease in all genotype groups with differing incubation periods, although M112T and L141F polymorphisms seemed to give some protection. Scrapie isolate SSBP/1, which has the shortest incubation period in sheep with at least one VRQ PRNP allele, also produced disease following sub-cutaneous inoculation in ARQ/ARQ animals of New Zealand origin, but ARQ/ARQ sheep from the Roslin flock survived the challenge. Our results demonstrate that the links between PRNP genotype and clinical prion disease in sheep are much less secure than previously thought, and may break down when, for example, a different breed of sheep is moved into a new flock.