Prion protein polymorphisms in autochthonous European sheep breeds in respect to scrapie eradication in affected flocks

Available online at www.sciencedirect.com

Small Ruminant Research 75 (2008) 43–47

Prion protein polymorphisms in autochthonous European sheep breeds in respect to scrapie eradication in affected flocks Gesine L¨uhken ∗ , Shirin Lipsky, Christina Peter, Georg Erhardt Department of Animal Breeding and Genetics, Justus-Liebig University of Giessen, Ludwigstrasse 21B, D-35390 Giessen, Germany Received 22 June 2006; received in revised form 21 June 2007; accepted 24 July 2007 Available online 17 September 2007

Abstract Resistance or susceptibility of sheep to scrapie is influenced by polymorphisms in the prion protein (PrP) gene (PRNP). Consequently, scrapie eradication in affected sheep flocks within the European Community (EC) aims at quickly removing the highly susceptible haplotype for classical scrapie, V136 R154 Q171 (VRQ), and increasing the number of sheep with the most resistant genotype for classical scrapie, ARR/ARR. In total, 1672 blood samples of 56 sheep breeds originating from 15 European and Middle Eastern countries were analyzed for nucleotide polymorphisms at codons 136, 154 and 171 of PRNP using improved restriction-fragmentlength-polymorphism analyses. The frequency of the genotype ARR/ARR ranged from 0 to 42%. This genotype was absent in the samples from 11 breeds. Genotypes with at least one VRQ haplotype but no ARR haplotype showed frequencies from 0 to 21%. Such genotypes were not found in 34 of the 56 investigated breeds. The observed differences in the frequencies of PrP haplotypes and genotypes would have different consequences regarding culling of sheep in respect to genetically based scrapie eradication in affected flocks of these breeds. Furthermore, the rare ARK haplotype has been described for the first time in the Italian Bergamasca breed. © 2007 Elsevier B.V. All rights reserved. Keywords: Sheep; Scrapie; Prion protein; Polymorphisms; PrP genotyping

1. Introduction Within the European Community (EC) as well as in other countries, the present strategy of eradicating transmissible spongiform encephalopathies (TSE) in small ruminants is based on studies showing that the resistance or susceptibility of sheep to scrapie is influenced by polymorphisms in the prion protein (PrP) gene (PRNP). Resistance or susceptibility is strongly linked to variations at codons 136, 154 and 171 with ∗

Corresponding author. Tel.: +49 641 9937672; fax: +49 641 9937629. E-mail address: [email protected] (G. L¨uhken). 0921-4488/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2007.07.010

the five haplotypes A136 R154 Q171 (ARQ), A136 R154 R171 (ARR), A136 H154 Q171 (AHQ), A136 R154 H171 (ARH) and V136 R154 Q171 (VRQ) (Hunter, 1997). Consequently, PrP genotypes had been divided into risk groups and used in breeding programmes. The application of the European Community regulation No. 260/2003 to scrapie-affected sheep flocks aims at removing the VRQ haplotype and increasing the number of sheep with the genotype ARR/ARR as soon as possible, with respect to the present frequency of the ARR haplotype within the affected flock and prevention of inbreeding. Apart from the five PrP haplotypes listed above, rare haplotypes such as ARK, TRQ, AHR and VRR have been identified (Kutzer et al., 2002; Gombojav et al., 2003; Guo et al., 2003; Billinis et al., 2004), again correspond-

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ing to amino acid polymorphisms at codons 136, 154 and 171. These haplotypes are presently not determined by some of the routinely applied methods for genotyping ovine PrP. The haplotype ARK may be restricted to some groups of sheep breeds, as it has only been described in Asian fat tail sheep (Gombojav et al., 2003; Zhang et al., 2004), in crossbred Greek sheep (Billinis et al., 2004), in Xinjiang local sheep breeds in China (Lan et al., 2006), in hair sheep (Guo et al., 2003) and in some breeds belonging to the mountain group, like Italian Biellese (Acutis et al., 2004), German Krainer Steinschaf and Weisses Bergschaf (G. L¨uhken, personal communication). Presently, the ARK haplotype has been identified in only one scrapie-positive sheep with the PrP genotype ARK/ARH (Acutis et al., 2006). Therefore lysine at codon 171 does not confer full resistance, but its impact on susceptibility to ovine scrapie is still unknown. The aim of this study was to evaluate frequencies of PrP polymorphisms at codons 136 (A/V), 154 (R/H) and 171 (Q/R/H) in autochthonous sheep breeds from Europe and the Middle East. Additionally, we determined the frequency of the rare PrP haplotype ARK in breeds in which this haplotype has previously been described and/or in breeds deriving from such (Waßmuth et al., 2001). 2. Materials and methods Within the ECONOGENE project (http://lasig.epfl.ch/ projets/econogene/), 1672 blood samples of 56 sheep breeds originating from 15 European and Middle Eastern countries were randomly collected (Table 1). The breeds were mainly autochthonous with the exception of a few cosmopolitan Merino breeds. Sampling criteria were to collect per breed at most three unrelated animals (one male and two females) per flock from 11 registered breeding farms covering the traditional rearing area of the breed resulting in a total of 15–31 animals per breed (Marsan and ECONOGENE Consortium, 2005). The samples were analyzed for the amino acids encoded at positions 136 (A/V), 154 (R/H) and 171 (Q/R/H) of the PrP using restriction-fragment-length-polymorphism (RFLP) analysis as described before (L¨uhken et al., 2004). This method has been optimized by shortening the primers (forward primer: 5 GGCAGGAGCTGCTGCAGCT-3 , nucleotide 22622–22640 in GenBank sequence U67922; reverse primer for digestion with BspHI: 5 -CACAAAGTTGTTCTGGTTACTATC-3 , nucleotide 22791–22814 in GenBank sequence U67922; reverse primer for double digestion with BspHI and BspDI: 5 -CAAAGTTGTTCTGGTTACTATAT-3 , nucleotide 22790–22812 in GenBank sequence U67922) and modifying the PCR conditions in respect to MgCl2 concentration (2.5 mM) and annealing temperature (56 ◦ C).

The breed Weisses Bergschaf, its related breeds Braunes Bergschaf and Bergamasca and the breed Cikta, related to Krainer Steinschaf, have been screened for the amino acid lysine at codon 171 of PrP. For this purpose, a 545 bp PCRfragment containing codon 171 of the ovine PRNP was amplified in a 50 ␮l-reaction containing 15 pmol each of the forward primer 5 -AACCAACATGAAGCATGTGGC-3 (nucleotide 22604–22624 in GenBank sequence U67922) and the reverse primer 5 -AAGCAAGAAATGAGACACCACC3 (nucleotide 23127–23148 in GenBank sequence U67922), 10–50 ng of genomic DNA, 10 mM of each dNTP, 2.0 mM MgCl2 and 1.5 U Taq polymerase in onefold reaction buffer under the following cycling conditions: denaturation at 94 ◦ C for 1.5 min, 40 amplification cycles including denaturation at 94 ◦ C for 15 s, annealing at 60 ◦ C for 20 s and extension at 72 ◦ C for 45 s, followed by a final 5-min extension at 72◦ C. Digestion of this 545 bp fragment with the restriction enzyme Mbo I at 37 ◦ C for 3 h resulted in several fragments. A 379 bp fragment occurs with the lysine at codon 171 and two fragments of 182 bp and 197 bp can be observed in the presence of other haplotypes. These methods have been validated by sequencing and cloning procedures.

3. Results Thirty-eight of the investigated 56 breeds showed four or five different PrP haplotypes, while in Morkaraman, Rubia del Molar and Karakul, only two PrP haplotypes (ARR and ARQ) were observed (Table 1). ARR and ARQ haplotypes occurred in all breeds and together represented at least 51% of the PrP haplotypes in each breed (on average 86%). Haplotypes AHQ, VRQ and ARH were found in 44, 31 and 26 breeds, respectively. In Bergamasca, one sheep carried the rare ARK haplotype, therefore six PrP haplotypes occurred in this breed. The frequency of ARR, with the lowest value (8%) in Manchega, was highest in Exmoor Horn with 68%, while the frequency of ARQ, ranging from 23 to 87% in all other breeds, was by far the lowest in Exmoor Horn (less than 10%). Moreover, in the latter breed, the frequency of VRQ was the highest (18%), followed by Anogeiano with 16%. In 10 breeds, the percentage of AHQ exceeded 10%, with the highest frequency found in Skopelos (27%). The average frequency of ARH was 3% and was exceptionally high in Akkaraman (38%). The frequency of the ARR/ARR genotype was highest in Exmoor Horn (42%) and about 30% in Sfakia, Polish Merino, Piliorritiko, Laticauda, Kalarritiko and Anogeiano, while in 11 of the breeds, no ARR/ARR genotype was identified (Table 1). The combined frequencies of genotypes including at least one ARR and no VRQ haplotype varied widely from 16 to 84%. In Hungarian Tsiagia, the frequency of genotypes including at

Table 1 Breed names, countries of origin, sample numbers (n) and frequencies (%) of PrP haplotypes and genotypes Breed

n

Frequencies (%) of PrP haplotypes

Frequencies (%) of PrP genotypes

ARR

ARQ

AHQ

ARH

ARK

VRQ

TR IT GR AL IT DE PT HU ES TR IT UK IT PL DE

17 31 31 30 31 16 30 31 31 31 31 31 31 31 31

8.82 32.26 50.00 31.67 9.68 21.88 16.67 19.35 30.65 16.13 25.81 67.74 35.48 40.32 9.68

52.94 53.23 32.26 60.00 70.97 75.00 76.67 67.74 67.74 80.65 62.90 9.68 53.23 53.23 80.65

0.00 8.06 1.61 5.00 4.84 0.00 3.33 12.90 0.00 3.23 4.84 4.84 8.06 6.45 9.68

38.24 6.45 0.00 0.00 4.84 0.00 3.33 0.00 0.00 0.00 6.45 0.00 3.23 0.00 0.00

0.00 0.00 0.00 0.00 1.61 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 16.13 3.33 8.06 3.13 0.00 0.00 1.61 0.00 0.00 17.74 0.00 0.00 0.00

ARR/ ARR 0.00 0.00 29.03 13.33 0.00 0.00 0.00 6.45 12.90 6.45 9.68 41.94 9.68 12.90 0.00

ARR/ ARQ 11.76 48.39 16.13 30.00 16.13 43.75 30.00 22.58 32.26 19.35 25.81 12.90 45.16 51.61 16.13

ARR/ AHQ 0.00 6.45 0.00 0.00 0.00 0.00 0.00 3.23 0.00 0.00 0.00 6.45 0.00 3.23 3.23

ARR/ ARH 5.88 9.68 0.00 0.00 0.00 0.00 3.33 0.00 0.00 0.00 6.45 0.00 6.45 0.00 0.00

SA HU HU GR PL GR RO TR GR GR IT GR ES DE TR SA SA GR EG GR PL PL HU DE RO RO ES AL UK ES GR AL GR ES UK FR RO DE UK PL PL

29 31 29 31 31 30 31 30 31 31 31 31 31 28 31 29 31 30 31 31 31 30 30 31 31 31 29 31 31 31 31 31 31 31 31 31 31 15 31 31 31

18.97 32.26 32.76 43.55 51.61 43.33 12.90 25.00 22.58 46.77 51.61 24.19 8.06 16.07 22.58 18.97 30.65 40.00 24.19 53.23 58.06 40.00 28.33 45.16 41.94 43.55 22.41 32.26 35.48 24.19 51.61 41.67 22.58 22.58 50.00 14.52 37.10 10.00 46.77 40.32 46.77

77.59 59.68 53.45 48.39 35.48 50.00 87.10 73.33 62.90 29.03 37.10 59.68 87.10 69.64 77.42 75.86 62.90 50.00 61.29 35.48 40.32 40.00 45.00 38.71 50.00 50.00 77.59 48.39 54.84 69.35 37.10 41.67 29.03 70.97 22.58 80.65 54.84 70.00 43.55 40.32 43.55

0.00 4.84 3.45 6.45 3.23 3.33 0.00 0.00 9.68 22.58 3.23 6.45 3.23 14.29 0.00 0.00 0.00 10.00 3.23 6.45 0.00 6.67 23.33 0.00 0.00 3.23 0.00 8.06 8.06 3.23 4.84 8.33 27.42 6.45 24.19 1.61 1.61 6.67 8.06 19.35 4.84

3.45 1.61 0.00 0.00 4.84 0.00 0.00 0.00 0.00 1.61 8.06 9.68 1.61 0.00 0.00 3.45 6.45 0.00 11.29 1.61 0.00 5.00 3.33 8.06 4.84 1.61 0.00 0.00 0.00 1.61 0.00 0.00 6.45 0.00 0.00 3.23 0.00 13.33 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 1.61 10.34 1.61 4.84 3.33 0.00 1.67 4.84 0.00 0.00 0.00 0.00 0.00 0.00 1.72 0.00 0.00 0.00 3.23 1.61 8.33 0.00 8.06 3.23 1.61 0.00 11.29 1.61 1.61 6.45 8.33 14.52 0.00 3.23 0.00 6.45 0.00 1.61 0.00 4.84

6.90 9.68 13.79 29.03 19.35 16.67 3.23 3.33 0.00 22.58 29.03 6.45 0.00 0.00 6.45 6.90 9.68 16.67 9.68 29.03 29.03 13.33 16.67 12.90 22.58 22.58 3.45 12.90 6.45 6.45 32.26 13.33 3.23 0.00 25.81 0.00 16.13 0.00 25.81 6.45 16.13

24.14 45.16 34.48 22.58 45.16 43.33 19.35 40.00 35.48 29.03 29.03 29.03 16.13 25.00 32.26 24.14 41.94 33.33 22.58 32.26 54.84 36.67 16.67 51.61 25.81 35.48 37.93 25.81 45.16 29.03 29.03 36.67 12.90 38.71 29.03 29.03 38.71 13.33 25.81 48.39 54.84

0.00 0.00 3.45 3.23 3.23 3.33 0.00 0.00 6.45 16.13 6.45 0.00 0.00 7.14 0.00 0.00 0.00 13.33 0.00 9.68 0.00 6.67 6.67 0.00 0.00 3.23 0.00 0.00 12.90 6.45 3.23 13.33 12.90 6.45 19.35 0.00 0.00 0.00 12.90 19.35 3.23

0.00 0.00 0.00 0.00 9.68 0.00 0.00 0.00 0.00 3.23 9.68 6.45 0.00 0.00 0.00 0.00 0.00 0.00 6.45 3.23 0.00 6.67 0.00 3.23 9.68 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 6.67 0.00 0.00 0.00

ARQ/ ARQ 23.53 25.81 19.35 40.00 54.84 50.00 56.67 45.16 51.61 70.97 45.16 0.00 25.81 22.58 67.74

AHQ/ ARQ 0.00 3.23 3.23 10.00 9.68 0.00 6.67 22.58 0.00 0.00 6.45 3.23 9.68 9.68 9.68

AHQ/ AHQ 0.00 3.23 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.23 0.00 0.00 3.23 0.00 3.23

AHQ/ ARH 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.23 0.00 0.00 0.00 0.00

62.07 29.03 24.14 32.26 9.68 26.67 77.42 53.33 38.71 6.45 19.35 32.26 77.42 50.00 61.29 58.62 35.48 33.33 38.71 16.13 12.90 16.67 26.67 6.45 35.48 29.03 58.62 22.58 29.03 51.61 19.35 16.67 9.68 48.39 3.23 64.52 32.26 53.33 29.03 9.68 9.68

0.00 9.68 3.45 9.68 3.23 3.33 0.00 0.00 6.45 16.13 0.00 12.90 3.23 14.29 0.00 0.00 0.00 0.00 6.45 3.23 0.00 6.67 16.67 0.00 0.00 3.23 0.00 16.13 3.23 0.00 3.23 3.33 12.90 6.45 9.68 0.00 3.23 6.67 3.23 12.90 6.45

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.23 6.45 0.00 0.00 0.00 3.57 0.00 0.00 0.00 3.33 0.00 0.00 0.00 0.00 10.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 9.68 0.00 6.45 0.00 0.00 0.00 0.00 3.23 0.00

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.23 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.33 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.23 0.00 6.67 0.00 0.00 0.00

ARQ/ ARH 47.06 3.23 0.00 0.00 3.23 0.00 3.33 0.00 0.00 0.00 3.23 0.00 0.00 0.00 0.00 6.90 3.23 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 6.45 12.90 0.00 0.00 0.00 6.90 12.90 0.00 16.13 0.00 0.00 0.00 3.33 6.45 0.00 3.23 0.00 0.00 0.00 3.23 0.00 0.00 9.68 0.00 0.00 3.23 0.00 13.33 0.00 0.00 0.00

ARH/ ARH 11.76 0.00 0.00 0.00 3.23 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

ARQ/ ARK 0.00 0.00 0.00 0.00 3.23 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

ARR/ VRQ 0.00 0.00 25.81 6.67 3.23 0.00 0.00 0.00 3.23 0.00 0.00 32.26 0.00 0.00 0.00

ARQ/ VRQ 0.00 0.00 6.45 0.00 0.00 6.25 0.00 0.00 0.00 0.00 0.00 3.23 0.00 0.00 0.00

AHQ/ VRQ 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.23 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 3.23 6.45 6.67 0.00 3.33 3.23 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.23 3.23 3.33 0.00 9.68 3.23 3.23 0.00 12.90 0.00 0.00 6.45 6.67 12.90 0.00 0.00 0.00 3.23 0.00 3.23 0.00 3.23

0.00 3.23 20.69 0.00 3.23 0.00 0.00 0.00 6.45 0.00 0.00 0.00 0.00 0.00 0.00 3.45 0.00 0.00 0.00 3.23 0.00 3.33 0.00 6.45 3.23 0.00 0.00 9.68 3.23 3.23 3.23 10.00 3.23 0.00 0.00 0.00 3.23 0.00 0.00 0.00 6.45

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.23 0.00 9.68 0.00 6.45 0.00 0.00 0.00 0.00 0.00 0.00

ARH/ VRQ 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

VRQ/ VRQ 0.00 0.00 0.00 0.00 6.45 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.33 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.23 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.33 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.23 0.00 0.00 0.00 0.00

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Akkaraman Altamurana Anogeiano Bardhoka Bergamasca Braunes Bergschaf Churra Cikta Colmenare˜na Daglic¸ Delle Langhe Exmoor Horn Gentile di Puglia Gorska Graue Geh¨ornte Heidschnucke Heri Hungarian Merino Hungarian Tsiagia Kalarritiko Kamieniec Karagouniko Karakul Karayaka Kefallinias Kymi Laticauda Lesvos Manchega Merinolandschaf Morkaraman Naemi Najdi Orino Ossimi Piliorritiko Polish Merino Pomorska Racka Rhoenschaf Romanian Merino Romanian Tsigaia Rubia del Molar Ruda Scottish Blackface Segure˜na Sfakia Shkodrane Skopelos Spanish Merino Swaledale Thˆone et Martod Turcana Weisses Bergschaf Welsh Mountain Wrzosowka Zelazna

Countrya

a AL, Albania; DE, Germany; EG, Egypt; ES, Spain; FR, France; GR, Greece; HU, Hungary; IT, Italy; PL, Poland; PT, Portugal; RO, Romania; SA, Saudi Arabia; TR, Turkey; UK, United Kingdom.

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least one VRQ and no ARR haplotype was extraordinarily high (21%), followed by Skopelos with 16% and Ruda, Shkodrane and Pomorska with 10%. However, in 34 breeds no sample had a genotyping result belonging to this combination of PrP haplotypes. Among the breeds with PrP genotypes including at least one VRQ haplotype, Rhoenschaf, Shkodrane, Exmoor Horn, Hungarian Tsigai, Anogeiano, Ruda and Skopelos showed a combined frequency higher than 15%. Approximately one third of the sampled sheep from Exmoor Horn, Anogeiano and Skopelos had PrP genotypes belonging to these PrP genotypes. 4. Discussion For 37 of the investigated sheep breeds, this is to our knowledge the first report of PrP haplotype and genotype frequencies. These include breeds from Albania, Egypt, Greece (apart from Karagouniko), Hungary, Poland, Romania, Saudi-Arabia, Spain (apart from Manchega and Spanish Merino) and Turkey. Forty-one of the 56 breeds analyzed in this study were sampled in European Community Member States, where regulation No. 260/2003 of the European Community currently is applied in the case of ovine scrapie. According to this regulation, all breeding rams remaining within the scrapie-affected flock have to carry the PrP genotype ARR/ARR, while all ewes which carry at least one ARR haplotype and no VRQ haplotype can be maintained for breeding. Considering the combined frequencies of PrP genotypes with at least one ARR haplotype and no VRQ haplotype of the breeds investigated, applying this regulation to scrapie-affected purebred flocks of these, in many cases rare, breeds would result in the culling “only” 16 but up to 84% of ewes. Only in the two Polish breeds Polish Merino and Kamieniec could more than 75% of the ewes remain within a scrapie-affected purebred flock. In 25 of the investigated breeds, 25–50% of the ewes would have to be culled or slaughtered. The ARK haplotype has been described for the first time in the Bergamasca breed. The identification of only one out of 1672 sheep carrying the ARK haplotype supports its rarity and its occurrence in distinct groups of sheep breeds, in this case the mountain sheep group. The PrP haplotype and genotype frequencies obtained in this study will change in the future within most of the breeds, mainly due to breeding programs for scrapie resistance currently pursued in Member States of the European Community and in other countries, but also due to further selection activities. Furthermore, it should be taken into account that due to the limited sample numbers, minor changes in PrP haplotype and geno-

type frequencies might also occur if a larger number of animals per breed would be sampled. It has been observed recently that the PrP genotype ARR/ARR does not confer resistance and the PrP haplotype VRQ is not associated with a high susceptibility to the so-called atypical strains of scrapie (e.g. Moum et al., 2005; L¨uhken et al., 2007; Saunders et al., 2006). As a consequence, the lately adoped regulation No. 727/2007 of the European Community offers different policies for scrapie-affected sheep flocks on the basis of the scrapie type. However, breeding for the PrP haplotype ARR – while avoiding inbreeding – is the best way to omit culling of sheep in the case of classical ovine scrapie. 5. Conclusion The varying frequencies of the PrP haplotypes and genotypes investigated in 56 European and Middle Eastern sheep breeds reflect a different situation for each sheep breed with respect to the scrapie eradication strategy currently applied in the European Community. In the case that PrP genotyping and selection as prescribed in regulation No. 260/2003 (EC) would be executed in scrapie-affected purebred flocks of these breeds, between 16 and 84% of the ewes would have to be culled, resulting in a substantial loss of genetic and therefore breeding material. The rare PrP haplotype ARK was found only in one animal belonging to the Bergamasca breed. Acknowledgements Part of this study was funded within the ECONOGENE project (http://lasig.epfl.ch/projets/econogene/). We gratefully thank Gottfried Brem (AGROBIOGEN, Hilgertshausen, Germany) for providing ovine DNA standard samples including the PrP allele ARK. References Acutis, P.L., Sbaiz, L., Verburg, F., Riina, M.V., Ru, G., Moda, G., Caramelli, M., Bossers, A., 2004. Low frequency of the scrapie resistance-associated allele and presence of lysine-171 allele of the prion protein gene in Italian Biellese ovine breed. J. Gen. Virol. 85, 3165–3172. Acutis, P.L., Martucci, F., Mazza, M., Peletto, S., Iulini, B., Corona, C., Bozzetta, E., Casalone, C., Caramelli, M., 2006. A case of scrapie in a sheep carrying the lysine-171 allele of the prion protein gene. Arch. Virol. 151, 1875–1880. Billinis, C., Psychas, V., Leontides, L., Spyrou, V., Argyroudis, S., Vlemmas, I., Leontides, S., Sklaviadis, T., Papadopoulos, O., 2004. Prion protein gene polymorphisms in healthy and scrapie-affected sheep in Greece. J. Gen. Virol. 85, 547–554.

G. L¨uhken et al. / Small Ruminant Research 75 (2008) 43–47 Gombojav, A., Ishiguro, N., Horiuchi, M., Serjmyadag, D., Byambaa, B., Shinagawa, M., 2003. Amino acid polymorphisms of PrP gene in Mongolian sheep. J. Vet. Med. Sci. 65, 75–81. Guo, X., Kupfer, D.M., Fitch, G.Q., Roe, B.A., Desilva, U., 2003. Identification of a novel lysine-171 allele in the ovine prion protein (PRNP) gene. Arch. Virol. 34, 303–305. Hunter, N., 1997. PrP genetics in sheep and the applications for scrapie and BSE. Trends Microbiol. 5, 331–334. Kutzer, T., Pfeiffer, I., Brenig, B., 2002. Identification of new allelic variants in the ovine prion protein (PrP) gene. J. Anim. Breed. Genet. 119, 201–208. Lan, Z., Wang, Z.L., Liu, Y., Zhang, X., 2006. Prion protein gene (PRNP) polymorphisms in Xinjiang local sheep breeds in China. Arch. Virol. 151, 2095–2101. L¨uhken, G., Buschmann, A., Groschup, M.H., Erhardt, G., 2004. Prion protein allele A136H154Q171 is associated with high susceptibility to scrapie in purebred and crossbred German Merinoland sheep. Arch. Virol. 149, 1571–1580. L¨uhken, G., Buschmann, A., Brandt, H., Eiden, M., Groschup, M.H., Erhardt, G., 2007. Epidemiological and genetical differences between classical and atypical scrapie cases. Vet. Res. 38, 65–80.

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