Amino acid polymorphisms of PrP gene in Mongolian sheep

To characterize amino acid polymorphisms in sheep prion protein (PrP), we analyzed the PrP genes from 271 sheep of 4 breeds (Khalkh, Yeroo, Orkhon and Khangai) raised in central Mongolia (Tuv, Uvurkhangai and Selenge prefectures). A total of 16 genotypes and 8 allelic variants of the PrP gene at codons 112, 136, 154 and 171 were found. At codon 171, 1.8% of the sheep had arginine/arginine (R/R) (resistant to scrapie) and 66.8% had glutamine/glutamine (Q/Q) (susceptible to scrapie). Several Yeroo and Orkhon sheep raised in Selenge prefecture had valine at codon 136 (136V) (highly susceptible to scrapie). Several Yeroo, Orkhon and Khangai sheep raised in Selenge prefecture had histidine at codon 154 (154H). Novel polymorphisms of valine (V) and serine (S) at codon 127, lysine (K) at codon 171, and leucine (L) and arginine (R) at codon 189 were also found in Khalkh, Yeroo and Orkhon sheep. It is not known whether these novel polymorphisms affect scrapie susceptibility.     

Natural scrapie in British sheep: breeds, ages and PrP gene polymorphisms.

One hundred and sixty-seven sheep of 32 breeds and crossbreeds affected by natural scrapie throughout Britain were tested for the presence of restriction fragment length polymorphisms of the PrP gene observed when their DNA was digested with EcoRI or HindIII. These polymorphisms have already been associated with different susceptibilities to experimental scrapie (controlled by alleles of the Sip gene) in a flock of Cheviot sheep. In two studies 86 to 92 per cent of the sheep were found to carry the PrP gene EcoRI fragment e1 which is associated with high susceptibility (or the sA allele of Sip) to experimental scrapie. The PrP gene HindIII genotypes of the natural scrapie sheep were not apparently associated with differences in susceptibility to scrapie. There was no link between the polymorphisms and the age or breed of the affected sheep.     

Identification of new allelic variants in the ovine prion protein (PrP) gene

The association between scrapie and polymorphisms of the prion protein (PrP) gene was studied in 1108 German sheep of 33 different breeds. The aim of the investigation was the determination of the codons 136, 154 and 171 of the PrP gene, which are important for scrapie susceptibility. In addition to the published allelic variants ARR, ARQ, AHQ, ARH and VRQ, two novel, rare haplotypes (AHR and VRR) were found in the breeds of Texel, Nolana and Suffolk. A comparison of PrP genotype frequencies among the analysed different breeds revealed distinct variations. Breeds such as Texel showed a complex genotype distribution over 17 variants, while breeds such as Friesian Milk Sheep indicated only seven different genotypes.     

PrP genotype frequencies in German breeding sheep and the potential to breed for resistance to scrapie

Genetic susceptibility to scrapie is associated with polymorphisms in three different codons of the ovine prion protein (PrP) gene (136, 154, 171). Studies of PrP genotypes linked to scrapie have revealed the resistance of homozygous PrPARR/PrPARR animals and the high risk of PrPVRQ/PrPVRQ and PrPvRQ/PrPARQ animals in scrapie-affected flocks. The selection of PrPARR/PrPARR genotypes may therefore provide a strategy for controlling clinical scrapie. The genotypes of 1361 German breeding sheep from 15 different breeds in northern Germany were determined. Apart from the wildtype allele PrPARQ, at least four mutually exclusive allelic variants were found. The greatest variability within the PrP gene was encountered in texel sheep, in which 14 PrP genotypes were found. In the important meat breeds, Suffolk, German whiteheaded mutton and German blackheaded mutton, the PrPARR allele was predominant, and in these breeds the breeding of scrapie-resistant pedigree flocks within four generations seems to be a feasible option. In the texel sheep, the German merino, the German milk and the German land sheep breeds, the frequency of the PrPARR allele was much lower, and in several breeds no homozygous rams were available for breeding purposes. In these breeds the breeding strategy would depend on the number of heterozygous rams available, but resistant pedigree flocks could be achieved within nine generations.     

PrP polymorphisms in Basque sheep breeds determined by PCR-restriction fragment length polymorphism and real-time PCR

Two new PCR-based methods were developed to decode prion protein (PrP) gene polymorphisms at codons 136, 154 and 171: a PCR-restriction fragment length polymorphism (RFLP) analysis consisting of two PCR reactions followed by three enzymatic digestions, and a real-time PCR consisting of four reactions with seven fluorogenic probes. Both methods were used to study the distribution of PrP gene polymorphisms in a representative sample (1297 animals) of the populations of the two native breeds of sheep of the Spanish Basque Country, Latxa and Carranzana. Fourteen genotypes were found in the Latxa breed, in which ARQ/ARQ was the genotype most frequently observed (49.3 per cent), followed by ARR/ARQ (32.6 per cent) and ARQ/ARH (5.8 per cent). The genotype associated with the highest resistance to scrapie (ARR/ARR) was present in 5 per cent of the animals analysed. Similar results were observed in the Carranzana sheep.     

PrP genotype frequencies of Quebec sheep breeds determined by real-time PCR and molecular beacons

The allele and genotype frequencies of the prion protein gene (PrP), known to have an impact on scrapie susceptibility, were determined by real-time PCR for 500 Quebec purebred rams. Molecular beacons were very efficient in discriminating the 5 alleles investigated. Polymorphisms at coding positions 136, 154, and 171 of the PrP gene were analyzed using 3 separate real-time PCR reactions and a total of 7 molecular beacons. A total of 4 different alleles (ARQ, ARR, AHR, and VRQ) were observed at different frequencies among the 7 breeds of sheep investigated. Results show that more than 50% of the rams in every breed carried at least one ARR allele, which is considered the most resistant to scrapie. The susceptibility ARQ allele was also present in every breed and together with the ARR allele, they were the most frequent alleles found in Quebec rams. The VRQ allele associated with the highest susceptibility to scrapie occurred in 5 of the 7 breeds, although at low frequencies. Overall, the results indicate that the frequencies of PrP alleles and genotypes in common breeds of sheep in Quebec make it feasible to reduce scrapie risk by selective breeding.     

Influence of prion protein gene polymorphisms on performance traits in German meat sheep breeds

PrP polymorphisms influence the scrapie susceptiblility of sheep. The objective of this study was to analyse the association between performance traits and the PrP genotype in the sheep breeds German black-headed and German white-headed mutton, Bleu du Maine, German mutton merino, Leine, Texel and Suffolk from Lower Saxony and Westphalia. We analysed performance traits such as scores for muscle mass, type and wool quality and the calculated daily weight gain using linear animal models. In all seven breeds no statistically significant associations were found between performance traits and the occurrence of ARR alleles, and the ARR/ARR genotypes, respectively. All genotyped sheep of all breeds investigated showed significantly superior performance traits in comparison to the non-genotyped animals.     

新疆地方绵羊品种PRNP基因多态性研究

从新疆采取了8个地方绵羊品种的血液样品171份,提取绵羊基因组DNA,用PCR方法扩增绵羊PRNP基因,通过序列测定,对它们的PRNP基因型进行研究,确定了PRNP基因136、154、171位密码子的多态性为136(A/A),154(H/R)和171(Q/R/H/K),结果发现所检测的新疆地方绵羊品种PRNP基因136位密码子均为A,其基因型均为A型痒病抵抗性基因型。     

Protective effect of the AT137RQ and ARQK176 PrP alleles against classical scrapie in Sarda breed sheep

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), 154 (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 the USA to increase the frequency of the resistant ARR allele in sheep populations. Herein, we report the results of a multi-flock study showing the protective effect of polymorphisms other than those at codons 136, 154 and 171 in Sarda breed sheep. All ARQ/ARQ affected sheep (n = 154) and 378 negative ARQ/ARQ controls from four scrapie outbreaks were submitted to sequencing of the PrP 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 AT₁₃₇RQ and ARQK₁₇₆ 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.     

Clinical signs, histopathology and genetics of experimental transmission of BSE and natural scrapie to sheep and goats

This paper compares the dinical signs, histopathology, detection of PrPSc protein and PrP genetics of the transmission of BSE to sheep and goats, with the effects of the transmission of natural scrapie from a brain homogenate from a single sheep. After intracerebral and oral inoculations there were similarities in the clinical signs due to the two sources of infection, but there were differences in pathology at the end stage of disease and in the genotypes of the sheep which succumbed to the challenges. The incubation period of BSE was associated with the sheep PrP codon 171 genotype, but the natural scrapie source, despite inducing disease only in known susceptible genotypes, showed no clear association with PrP genotype.     

Detection of prion protein gene polymorphisms in Baltic breeds of sheep

A total of 167 sheep belonging to the Estonian whiteheaded mutton, Estonian blackheaded mutton, Lithuanian coarsewool native, Lithuanian blackface and Latvian darkheaded mutton breeds, and a population of sheep kept isolated on the Estonian island of Ruhnu, were sequence-analysed for polymorphisms in the prion protein (PrP) gene, to determine their genotype and the allele frequencies of polymorphisms in PrP known to confer resistance to scrapie. A 939 base pair fragment of exon 3 from the PrP gene was amplified by pcr and analysed by direct sequencing. For animals showing polymorphism at two nucleotide positions, both haplotypes of these double-heterozygous genotypes were further verified by pcr cloning and sequence analysis. Known polymorphisms were observed at codons 136, 154 and 171, and six different haplotypes (arr, ahq, arh, ahr, arq and vrq) were determined. On the basis of these polymorphisms, the six populations of sheep possessed the resistant arr haplotype at different frequencies. The high-risk arq haplotype occurred in high frequencies in all six populations, but vrq, the haplotype carrying the highest risk, occurred at low frequencies and in only three of the populations.     

Linkage of the gene for the scrapie-associated fibril protein (PrP) to the Sip gene in Cheviot sheep

The gene Sip with two alleles, sA and pA, is the major gene determining the incubation period of scrapie in its natural host, sheep. Two lines of Cheviot sheep have been bred which differ in their response to experimental infection with SSBP/1 scrapie. The negative line have a decreased incidence of disease caused by SSBP/1 and are SippApA. The positive line have an increased incidence of disease and the majority are either SipsAsA or SipsApA; it is not possible to distinguish between the two genotypes on the basis of scrapie incubation time because the sA allele is fully dominant with SSBP/1 scrapie. There are also rare SippApA segregants in the positive line. The major protein (PrP) of scrapie-associated fibrils is encoded by a cellular gene and a cDNA copy of the hamster PrP mRNA has been used to analyse the restriction fragment length polymorphism of the two lines of Cheviot sheep. Two polymorphisms of the sheep PrP gene were found, by using HindIII and EcoRI, which appear to act as markers for the alleles of Sip. Using these polymorphisms it is now possible to assign a Sip genotype to the sheep in the Cheviot flock. Preliminary results from Anglo-Nubian goats and a cow are also reported.     

Frequencies of PrP genotypes in 38 breeds of sheep sampled in the National Scrapie Plan for Great Britain

Between October 2001 and January 2003 the prion protein (PrP) genotypes of over 250,000 sheep were determined through the operation of the National Scrapie Plan (NSP); the results for 38 breeds were analysed to provide an estimate of the underlying PrP genotype distribution of the British sheep population. Although there was marked variability among the genotype profiles of the different breeds, several trends emerged. A comparison of the allele frequencies demonstrated that the breeds could be grouped into three categories: breeds dominated by ARR and ARQ in which the frequency of ARR exceeded the frequency of ARQ; breeds dominated by ARR and ARQ in which the frequency of ARQ exceeded the frequency of ARR; and breeds with significant levels of either AHQ, ARH or VRQ. Hill breeds were more likely to have a lower proportion of animals at low risk of scrapie (NSP type 1) and a higher proportion of animals at an intermediate risk of scrapie (NSP type 3) than other breeds. Most breeds had a small proportion of animals at high risk of scrapie (NSP type 5). The frequency of ARR/VRQ (NSP type 4) was variable.     

Biology of PrPsc accumulation in two natural scrapie-infected sheep flocks.

Sheep scrapie is a prion disease that requires interaction of exogenous prions with host prion protein (PrP) supporting prion formation. Disease is associated with deposition of a host-generated conformational variant of PrP, PrPsc, in a variety of tissues, including brain, resulting in fatal spongiform encephalopathy. Efficiency of PrPsc formation is determined by polymorphisms in the PrP-coding sequence. This article adds to previous data of natural sheep scrapie, concentrating on the effect of host genotype and age on PrPsc accumulation patterns during preclinical and clinical disease. Two entire scrapie-infected, predominantly Suffolk-cross, sheep flocks euthanized for regulatory purposes were genotyped and analyzed for PrPsc deposition in various tissues using single- and dual-label immunohistochemistry. Scrapie, as defined by PrPsc deposition, occurred in 13/80 sheep. Preclinical disease was evident in nearly 70% of infected sheep, ranging in age from 14 months to 7 years. PrPsc accumulated systemically in the nervous tissue, various lymphoid tissues, both alimentary tract related and non-alimentary tract related, and the placenta. Clinical neurological illness was always associated with spongiform encephalopathy and PrPsc deposition in the brain. Only 6 of 9 sheep with preclinical scrapie had PrPsc deposition in the brain but widespread PrPsc deposition in peripheral lymphoid tissue, supporting previous data showing peripheral PrPsc accumulation preceding deposition in the brain. PrPsc colocalized with a marker for follicular dendritic cells throughout the lymphoid system. PrPsc also accumulated in the peripheral nervous system, particularly the nervous supply of the gastrointestinal tract. Abundant PrPsc was evident in trophoblast cells of placentomes but not in the endometrium, myometrium, or associated nervous plexus. PrPsc deposits were not observed in the mammary parenchyma or bone marrow. Scrapie susceptibility was defined genetically by PrP codon 171: PrPsc deposition was restricted to PrP genotype AA136RR154QQ171 in 12/13 cases or AV136RR154QQ171 in 1/13 cases. The earliest accumulation was observed in the single VRQ/ARQ heterozygous animal, consistent with the reported high scrapie susceptibility and brief incubation period observed in breeds with predominance of the V136R154Q171 allele. Disease occurred within, as well as independent of, mother-daughter lines, suggesting both maternal and nonmaternal transmission in the flocks.     

Identification of a new leucine haplotype (ALQ) at codon 154 in the ovine prion protein gene in Spanish sheep

Genetic susceptibility to scrapie is closely linked to variations at codons 136, 154, and 171 of the prion protein (PRNP) gene. This association between the PRNP genotype and susceptibility to scrapie is the basis of breeding programs for scrapie resistance in different countries. In this paper, we describe the method used with 2 Spanish dairy sheep breeds (Churra and Castellana) to ascertain the initial status of protection against scrapie as a first step toward adapting their breeding schemes to include resistance as a complementary selection criterion. The procedure for genotype identification is based on multiplex minisequencing methodology and has been shown to be accurate, easy to interpret, and to have a medium throughput. The frequency of the ARQ allele was similar in the 2 populations at nearly 70%. The ARR allele, associated with resistance in the homozygous state, reaches around 23% in Churras and nearly 20% in Castellanas. The high-risk VRQ allele appeared at a relatively low frequency in both breeds. No other haplotypes were found in these 2 breeds. Furthermore, in this screening we found a new allele carrying leucine at codon 154. This new genetic variant might play a role in susceptibility to scrapie because codon 154 belongs to a region considered to have an important role in conformational conversion of the cellular to the pathogenic protein.     

Prion protein gene polymorphism in four West African sheep populations

A total of 162 individuals, belonging to three Burkinabé and one Niger sheep populations, were analysed for prion protein (PrP) gene polymorphism at codons 136, 154 and 171. The ARQ allele was the most frequent in both the Burkinabé (86.7%) and the Niger (67.5%) sheep populations. The highly sensitive allele VRQ was not found in the sampled individuals. The highly resistant ARR allele was in very low frequency in the Burkina-Sahel (4.4%) and Mossi (3.2%) populations and was not present in the Djallonké and Touareg populations. Only 4 out of 15 possible PrP genotypes were identified in the sampled individuals. No favourable ARR/ARR genotypes were found in either of the breeds. Sequencing a subgroup of the samples allowed the identification of other five polymorphisms on the PrP gene sequence at codons 116, 138, 151, 237 and 240. The very low frequency of the ARR allele in the West African sheep should dissuade the implementation of a preventive selection programme aimed to increase resistance to scrapie, to avoid an extreme erosion of the genetic stock.     

Association between incubation time and genotype in sheep experimentally inoculated with scrapie-positive brain homogenate

OBJECTIVE: To compare incubation time and clinical signs of scrapie in codon 136/171 alanine-valine/glutamine-glutamine (AVQQ) experimentally inoculated sheep with that in sheep with the more common 136/171 AAQQ genotype. ANIMALS: 60 Suffolk sheep. PROCEDURE: Twenty-seven 171 QQ ewes purchased from 2 private flocks were bred with a 171 QQ Suffolk ram before being inoculated with a 20% solution of scrapie-positive brain homogenate (5 mL, PO) from sheep containing genotypes 136/154/171 AA/arginine-arginine (RR)/QQ, AVRRQQ, and VVRRQQ that had died of scrapie. Ewes had 33 lambs, which were inoculated in the same manner on the day of birth. RESULTS: All 16 genotype 136/154/171 AVRRQQ sheep that died of scrapie were 9 to 11 months of age; clinical signs lasted 1 day to 3 weeks with no wasting and only mild pruritus. The first AARRQQ sheep died with typical clinical signs of scrapie 27 months after inoculation, and 14 were still alive 37 to 42 months after inoculation. The 136/171 AVQQ sheep had minimal accumulation of modified cellular protein (PrP(SC)) as determined by immunohistochemical (IHC) staining within affected cells; thus the severity of clinical signs and time of death were not associated with brain lesions or the amount of PrP(SC) in brain TISSUE OF 136/154/171 AVRRQQ sheep as determined by IHC staining. CONCLUSIONS AND CLINICAL RELEVANCE: The rapid incubation time may have been influenced by the codon 136 genotype, a new unreported valine (V)-dependent strain of scrapie similar to strain SSBP/1, or the inoculum may have contained a traditional strain and a V-dependent or SSBP/1-like strain of scrapie.     

Detection of polymorphisms in the prion protein gene in a population of Irish Suffolk sheep

Natural scrapie is associated with polymorphisms in the prion protein (PrP) gene. In Suffolks, codon 171 is the codon at which most variation is found; RR171 is thought to be associated with resistance to developing the clinical signs of the disease and QQ171 is associated with susceptibility to the disease. The objectives of this study were first to determine the PrP genotypes of Suffolk stock rams in Ireland, and secondly to compare the genotype profiles of ram lambs from flocks where a breeding programme based on the genotype AA136RR154RR171 had been initiated and from flocks where there was no breeding programme based on PrP genotype. Approximately 13 per cent of the stock rams genotyped in the Irish population were genetically susceptible to showing the clinical signs of the disease. However, lambs from farms that had initiated a selective breeding strategy for RR171 over the past year had a larger proportion of RR171 and a smaller proportion of QQ171 than the stock rams or ram lambs from farms not applying a breeding strategy.     

Polymorphisms of caprine PrP gene detected in Japan

Polymorphism of the PrP gene is a primary factor influencing susceptibility and incubation period in natural and experimental scrapie in sheep and goats. Polymorphisms of the caprine PrP gene in Japan were examined in 118 goats. Eight allelic variants and 19 genotypes were obtained. Amino acid polymorphisms were observed at 7 codons: 102, 142, 143, 240, 127, 146 and 211 (the latter 3 are novel polymorphisms). The polymorphisms at codons 142M and 143R, which are associated with the resistance to scrapie, were relatively rare in the present study. Thus, the present results provide information about the caprine PrP gene that may be useful for assessing the risk of goat scrapie.