Ultrasensitive detection of scrapie prion protein derived from ARQ and AHQ homozygote sheep by interspecies in vitro amplification

Prions, infectious agents causing TSEs, are composed primarily of the pathogenic form (PrP(Sc)) of the PrP(C). The susceptibility of sheep to scrapie is determined by polymorphisms in the coding region of the PRNP, mainly at codons 136, 154, and 171. The efficiency of in vitro amplification of sheep PrP(Sc) seems to be linked also to the PrP genotype. PrP(Sc) derived from sheep with V(136)R(154)Q(171)-associated genotypes can be amplified efficiently by PMCA in the presence of additional polyanion such as poly A, but there are no reports that cite ultrasensitive detection of PrP(Sc) derived from sheep of other PrP genotypes. We report here that sheep PrP(Sc) derived from ARQ and AHQ homozygotes was amplified efficiently by serial PMCA using mouse brain homogenate as PrP(C) substrate. ARQ/ARQ PrP(Sc) was detected in infected brain homogenates diluted up to 10(-10) after five rounds of amplification, and AHQ/AHQ PrP(Sc) was detected in samples diluted up to 10(-8) after four rounds of amplification. On the other hand, amplification of PrP(Sc) from VRQ/ARQ sheep seemed to be less efficient under the experimental conditions used. The interspecies PMCA developed in this study may be useful in the detailed analysis of PrP(Sc) distribution in classical scrapie-infected ARQ and AHQ homozygote sheep.     

Associations of PrP genotype with lamb production traits in three commercial breeds of British hill sheep

The National Scrapie Plan (NSP) was launched in Great Britain in 2001, with the aim of eventually eradicating scrapie, a small ruminant transmissible spongiform encephalopathy, from the national sheep flock. Specifically, a selective breeding programme, the Ram Genotyping Scheme, was devised enabling pedigree ram breeders to reduce the number of scrapie-susceptible genotypes from their flocks. The effect of large-scale manipulation of PrP genotypes on commercially important traits within the sheep industry is, however, unknown. We have therefore examined production traits in a total of 43 968 lambs from 32 pedigree breeders across three British hill breeds, comprising 8163 North Country Cheviot (Hill), 21 366 Scottish Blackface and 14 439 Welsh Mountain lambs. Traits examined included: weights at birth, 8 and 20 weeks; ultrasonic fat and muscle depth, and average daily weight gain from 8 to 20 weeks. Linear mixed models were fitted for each trait, including animal (direct) genetic effects and up to three maternal effects. Potential associations with the PrP gene were assessed by fitting either PrP genotype or number of copies of individual alleles as fixed effects. A number of breed-specific significant associations between production traits and the PrP gene were found, but no consistent significant effects were detected across the three breeds. Breed-specific effects were as follows: (i) 0.37 kg higher birth weights (BWTs) in AHQ homozygous North Country Cheviot (Hill) lambs (P < 0.01); (ii) 0.16 kg higher BWTs in ARR homozygous Scottish Blackface lambs (P < 0.05); (iii) 0.5 kg higher 8-week weights in VRQ heterozygous Scottish Blackface lambs (P < 0.01); (iv) a 0.72 kg decrease in scan weight associated with homozygous ARR Welsh Mountain lambs (P < 0.01); (v) 0.51 mm higher ultrasonic muscle depths in AHQ homozygous Welsh Mountain lambs (P < 0.01); (vi) 0.48 mm lower ultrasonic muscle depths in Welsh Mountain lambs carrying one or more copies of the ARR allele (P < 0.05) and (vii) 0.2 mm higher ultrasonic fat depths in heterozygous VRQ Welsh Mountain lambs (P < 0.05). The use of a Bonferroni correction to define appropriate significance thresholds across the three datasets, which account for the large number of independent comparisons made, resulted in breed-specific comparisons, with P < 0.01 becoming significant at P0.05, and the remaining breed-specific comparisons no longer being significant. The absence of a common effect across the three breeds suggests that any true association found may be due to breed-specific alleles of neighbouring genes in linkage disequilibrium with the PrP locus.     

p.Asn176Lys and p.Met137Thr dimorphisms of the PRNP gene significantly decrease the susceptibility to classical scrapie in ARQ/ARQ sheep

In this study, we investigated the susceptibility to scrapie of Sarda breed sheep carrying the genotype ARQ/ARQ with additional polymorphisms at the PRNP gene. To do this, we examined 256 scrapie-affected sheep and 320 flock-mate negative controls from 24 flocks. Logistic regression analysis demonstrated that sheep carrying the ARQ/ARQ genotype with additional dimorphisms had lower risk of becoming scrapie affected when compared with those with ARQ/ARQ_wildtype genotype. ARQ/ARQ genotypes that were detected with heterozygous or homozygous p.Asn176Lys and p.Met137Thr dimorphisms were associated with the lowest susceptibility to the disease. A significant lower risk was also associated with the p.Arg154His dimorphism, while p.Leu141Phe had a protective effect that was not statistically significant.     

Prion protein gene (PrP) polymorphisms in healthy sheep in Turkey

Scrapie, a transmissible spongiform encephalopathy (TSE) or prion disease, is a fatal, neurodegenerative disease in sheep and goats. This disease has been known in Europe for more than 250 years. Susceptibility to scrapie is associated with polymorphisms in the sheep prion protein gene (PrP) gene. In sheep, polymorphism in the PrP gene has been identified at a number of codons, and polymorphisms at codons 136, 154 and 171 have reported linkage with susceptibility to scrapie. Polymorphisms at the PrP locus were studied in 413 animals representing three native sheep breeds (Imroz, Chios and Kıvırcık) in Turkey. Genomic DNA was obtained from blood, and genotypes were screened using PCR and direct DNA sequencing. We report 17 genotypes derived from seven different alleles. The most frequent genotype in the Kıvırcık sheep is ARQ/ARQ, whereas the ARR/ARQ genotype is predominant in the Chios and Imroz breeds. In general, the ARQ haplotype was the predominant haplotype. ARQ haplotype was also predominant in the Kıvırcık and Chios sheep breeds, whereas the Imroz sheep predominantly had the ARR haplotype. The susceptibility-associated VRQ haplotype was found in 2.38%, 0.35% and 0.81% of the Imroz, Kıvırcık and Chios sheep, respectively. Moreover, seven additional polymorphisms have been detected at codons G127S, G127V, H143R, G145S, Y172D, N174Y and Q189L. Among these polymorphisms, the N174Y allele is a novel polymorphism, and the G145S allele is a novel allele for a known polymorphic locus.     

Associations of PrP genotype with lamb production traits in three commercial breeds of British lowland sheep

The Ram Genotyping Scheme was launched in Great Britain in 2001 as part of the National Scrapie Plan and was devised to reduce and eventually eradicate classical scrapie susceptible genotypes from the national pedigree flock. Anecdotal claims from breeders suggest that sheep with more resistant PrP genotypes may have inferior phenotypes. In this study, we test this possibility for lamb production traits in three breeds of lowland sheep: Charollais (22 752 lambs), Poll Dorset (22 589 lambs) and Texel (23 492 lambs). Data were received from 50 breeders and comprised weights at birth, 8 weeks and scanning (from which average daily weight gain was derived), and ultrasonic muscle and fat depths. Animal (direct) genetic effects and up to three maternal effects were fitted in linear mixed models for each trait. Fitting either PrP genotype or number of copies of individual alleles carried as fixed effects allowed potential associations with the PrP gene to be assessed. There were no significant associations seen in the Poll Dorset breed; however, significant associations were found with the number of allele copies carried in the other two breeds included in this study. Charollais lambs carrying one copy of the VRQ allele had significantly (P < 0.01) greater ultrasonic muscle depth (0.58 mm) and fat depth (0.2 mm) than non-carriers. In the Texel breed, lambs with one ARR allele were significantly heavier than those with two or zero ARR alleles; heterozygous ARR lambs were 0.07 kg heavier at birth (P < 0.05), 0.42 kg heavier at 8 weeks (P < 0.01) and 0.17 kg heavier at scan weight (P < 0.01), than non-carriers. After Bonferroni corrections to adjust significance thresholds to account for the large number of independent comparisons made, all significant results remained so at P < 0.05 or greater, except for the ARR allele effect on birth weight in the Texel breed, which was no longer significant. These results compare favourably with others from studies on many continental breeds of sheep, published in recent years, and add credence to the conclusion that selection on PrP genotype is unlikely to have any noticeable impact on the measured growth and carcass traits in sheep.     

Two different scrapie prions isolated in Japanese sheep flocks

Two different scrapie prion strains with different characteristics were obtained from two sheep naturally infected with scrapie in Japan. In mice transmission, one (Tsukuba-1) showed shorter incubation periods (133+/-2 days) than the other (Tsukuba-2) (288+/-5 days). Spongiform changes and accumulation of an abnormal isoform of prion protein (PrP(Sc)) were observed throughout the brain in Tsukuba-1 inoculated mice, while the lesions and the PrP(Sc) accumulation were localized in the brain stem of mice with Tsukuba-2. Western blot analysis showed that there was no strain-specific glycoform of PrP(Sc) within these two strains. A super-infection experiment revealed that neither strain interfered with the other's propagation.     

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.     

Amyloidogenic unfolding intermediates differentiate sheep prion protein variants

Sheep is a unique example among mammalian species to present a strong correlation between genotype and prion disease susceptibility phenotype. Indeed a well-defined set of PrP polymorphisms at positions 136, 154 and 171 (sheep numbering) govern scrapie susceptibility, ranging from very high susceptibility for V136-R154-Q171 variant (VRQ) to resistance for A136-R154-R171 variant (ARR).To get better insight into the molecular mechanisms of scrapie susceptibility/resistance, the unfolding pathways of the different full-length recombinant sheep prion protein variants were analysed by differential scanning calorimetry in a wide range of pH. In the pH range 4.5-6.0, thermal unfolding occurs through a reversible one-step process while at pH <4.5 and >6.0 unfolding intermediates are formed, which are stable in the temperature range 65-80 degrees C. While these general behaviours are shared by all variants, VRQ and ARQ (susceptibility variants) show higher thermal stability than AHQ and ARR (resistance variants) and the formation of their unfolding intermediates requires higher activation energy than in the case of AHQ and ARR. Furthermore, secondary structures of the unfolding intermediates differentiate variants: ARR unfolding intermediate exhibits random coil structure, contrasting with the beta-sheet structure of VRQ and ARQ unfolding intermediates. The rate of the unfolding intermediate formation allows us to classify genetic variants along increasing scrapie susceptibility at pH 4.0, VRQ and ARQ rates being the highest. Rather poor correlation is observed at pH 7.2. Upon cooling, these intermediates refold into stable species, which are rich in beta-type secondary structures and, as revealed by thioflavin T fluorescence and electron microscopy, share amyloid characteristics. These results highlight the prion protein plasticity genetically modulated in sheep, and might provide a molecular basis for sheep predisposition to scrapie taking into account both thermodynamic stability and transconformation rate of prion protein.     

Prion gene (PRNP) haplotype variation in United States goat breeds (Open Access publication)

Scrapie eradication efforts cost 18 million dollars annually in the United States and rely heavily upon PRNP genotyping of sheep. Genetic resistance might reduce goat scrapie and limit the risk of goats serving as a scrapie reservoir, so PRNP coding sequences were examined from 446 goats of 10 breeds, 8 of which had not been previously examined at PRNP. The 10 observed alleles were all related to one of two central haplotypes by a single amino acid substitution. At least five of these alleles (M142, R143, S146, H154, and K222) have been associated with increased incubation time or decreased odds of scrapie. To the best of our knowledge, neither S146 nor K222 has been found in any goats with scrapie, though further evaluation will be required to demonstrate true resistance. S146 was more common, present in several breeds at widely varying frequencies, while K222 was observed only in two dairy breeds at low frequency. Overall, this study provides frequency data on PRNP alleles in US goats, shows the pattern of relationships between haplotypes, and demonstrates segregation of multiple scrapieassociated alleles in several breeds not examined before at PRNP.     

Relationship between γ‐interferon gene polymorphisms and susceptibility to brucellosis infection

Interferon‐gamma (IFN‐γ) is a pro‐inflammatory cytokine that plays a pivotal role in the defense mechanism against Brucella infection. It was hypothesized that the IFN‐γ in (+874 A/T in intron 1) TT and +5644 T/A, TT genotypes, which are reportedly associated with high IFN production, are associated with susceptibility to brucellosis in Iranian subjects. Genotyping of these IFN‐γ variants by an allele‐specific polymerase chain reaction method was performed in 281 subjects, comprising 153 patients with active brucellosis and 128 healthy controls. It was found that the +874 minor allele (A) and homozygote genotype (AA) were significantly more frequently present in brucellosis patients than in controls (OR = 2.588; 95% CI, 1.313–5.104; P = 0.006 for the AA genotype; OR = 1.575; 95% CI, 1.124–2.216; P = 0.010 for the A allele). However, the allelic and genotypic distribution of the IFN‐γ polymorphism at position UTR5644 A>T did not differ significantly between patients and controls (P > 0.05). The distribution of haplotypes in this study suggests that the T/A haplotype (+874/UTR5644), which was present more frequently in controls than in patients, may protect subjects against Brucella infection. It is suggested that IFN‐γ +874 AA genotype and A allele are risk factors for developing brucellosis infection in Iranian subjects.     

Different structural stability and toxicity of PrP(ARR) and PrP(ARQ) sheep prion protein variants

The polymorphisms at amino acid residues 136, 154, and 171 in ovine prion protein (PrP) have been associated with different susceptibility to scrapie: animals expressing PrP^ARQ [PrP(Ala136/Arg154/Gln171)] show vulnerability, whereas those that express PrP^ARR [PrP(Ala136/Arg154/Arg171)] are resistant to scrapie. The aim of this study was to evaluate the in vitro toxic effects of PrP^ARR and PrP^ARQ variants in relation with their structural characteristics. We show that both peptides cause cell death inducing apoptosis but, unexpectedly, the scrapie resistant PrP^ARR form was more toxic than the scrapie susceptible PrP^ARQ variant. Moreover, the α-helical conformation of PrP^ARR was less stable than that of PrP^ARQ and the structural determinants responsible of these different conformational stabilities were characterized by spectroscopic analysis. We observed that PrP toxicity was inversely related to protein structural stability, being the unfolded conformation more toxic than the native one. However, the PrP^ARQ variant displays a higher propensity to form large aggregates than PrP^ARR. Interestingly, in the presence of small amounts of PrP^ARR, PrP^ARQ aggregability was reduced to levels similar to that of PrP^ARR. Thus, in contrast to PrP^ARR toxicity, scrapie transmissibility seems to reside in the more stable conformation of PrP^ARQ that allows the formation of large amyloid fibrils.     

The effects of selective breeding against scrapie susceptibility on the genetic variability of the Latxa Black-Faced sheep breed

Breeding sheep populations for scrapie resistance could result in a loss of genetic variability. In this study, the effect on genetic variability of selection for increasing the ARR allele frequency was estimated in the Latxa breed. Two sources of information were used, pedigree and genetic polymorphisms (fifteen microsatellites). The results based on the genealogical information were conditioned by a low pedigree completeness level that revealed the interest of also using the information provided by the molecular markers. The overall results suggest that no great negative effect on genetic variability can be expected in the short time in the population analysed by selection of only ARR/ARR males. The estimated average relationship of ARR/ARR males with reproductive females was similar to that of all available males whatever its genotype: 0.010 vs. 0.012 for a genealogical relationship and 0.257 vs. 0.296 for molecular coancestry, respectively. However, selection of only ARR/ARR males implied important losses in founder animals (87 percent) and low frequency alleles (30 percent) in the ram population. The evaluation of mild selection strategies against scrapie susceptibility based on the use of some ARR heterozygous males was difficult because the genetic relationships estimated among animals differed when pedigree or molecular information was used, and the use of more molecular markers should be evaluated.     

Genetic linkage analysis between protein polymorphisms and the FecB major gene in sheep

A genetically linked marker locus is sought for the Booroola gene (FecB), a major gene which confers increased prolificacy in sheep. We examined 18 polymorphic proteins in sheep and found 10 to be informative in half-sib families where the Booroola gene was segregating. Recombination was observed between each of the protein loci and the Booroola gene. The loci and exclusion distance for each (calculated as the recombination fraction where the lod score was equal to -2.0) are as follows: NADH diaphorase, DIA1 (9.2 cM); arylesterase, EsA (11.9 cM); haemoglobin beta chain, HBB (17.5 cM); leucine amino peptidase, LAP (19.7 cM); malic enzyme, ME1 (14.8 cM); ovine plasminogen antigen, OPA (12.6 cM); alpha-1-protease inhibitor, PI2 (5.7 cM), erythrocyte 'X' protein, Prot-X (25.3 cM); post transferrin, PTF (2.2 cM); transferrin, TF (33.8 cM).     

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.     

Exposure of sheep scrapie brain homogenate to rumen-simulating conditions does not result in a reduction of PrP(Sc) levels

AIMS: Experiments were designed to evaluate the potential of rumen-simulating conditions to reduce PrP(Sc) levels. METHODS AND RESULTS: Scrapie-positive brain material was incubated under rumen-simulating conditions. Time points were taken over a 24-h period and PrP(Sc) levels were analysed by Western blot. No loss of PrP(Sc) was observed over a 24-h time period. CONCLUSIONS: Our results indicate that a fully developed rumen fermentation does not provide significant protection against prion infection via the oral route. Developmental changes including senescence of immune system function or other developmental changes in the gastrointestinal tract are potential mechanisms by which relative bovine spongiform encephalopathy (BSE) susceptibility might vary with age. SIGNIFICANCE AND IMPACT OF THE STUDY: Epidemiology of the BSE outbreak in the United Kingdom indicates that younger animals were at higher risk of infection. The rumen undergoes pronounced developmental changes early in life, coinciding with the introduction of fibre into the diet. The timeframe of highest risk of infection overlaps the time in life prior to full rumen development. This work indicates that a fully developed rumen does not provide significant protection against prion infection via the oral route of infection. This result implicates other developmental changes that are responsible for the age-dependent susceptibility of cattle to BSE.