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.

Protective effect of the AT137RQ and ARQK176 PrP allele 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), 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.