PRNP polymorphisms in four Italian sheep breeds

PRNP polymorphisms in four Italian sheep breeds

Livestock Science 181 (2015) 38–42 Contents lists available at ScienceDirect Livestock Science journal homepage: PRN...

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Livestock Science 181 (2015) 38–42

Contents lists available at ScienceDirect

Livestock Science journal homepage:

PRNP polymorphisms in four Italian sheep breeds Ludovica Curcio a,b, Carla Sebastiani a, Simone Ceccobelli b, Gabriele Vaccari c, Giovanni Pezzotti a, Emiliano Lasagna b,n, Massimo Biagetti a a

Area Ricerca e Sviluppo, Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche, Via G. Salvemini 1, 06126 Perugia, Italy Dipartimento di Scienze Agrarie, Alimentari e Ambientali, Università degli Studi di Perugia, Borgo XX giugno 74, 06121, Perugia, Italy c Dipartimento di Sanità Pubblica Veterinaria e Sicurezza Alimentare, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy b

art ic l e i nf o

a b s t r a c t

Article history: Received 27 February 2015 Received in revised form 15 September 2015 Accepted 2 October 2015

Prions are responsible for transmissible spongiform encephalopathies (TSEs), also called scrapie in sheep. Single nucleotide polymorphisms (SNPs) in PRNP have been shown to play a crucial role in terms of incubation period and/or susceptibility to scrapie. Considering codons 136, 154, 171, five main alleles, associated with different degrees of susceptibility to classical scrapie have been detected. Homozygous ARR sheep are resistant to scrapie infection, however this genotype is not so frequent in some breeds. VRQ and ARQ alleles on the other hand are associated with a high susceptibility to scrapie. Additional polymorphisms within the ARQ allele, have been associated with scrapie resistance. Therefore, it is important to assess the frequency of both main and minor allelic variants, in order to develop a breeding programme based on genetic selection that simultaneously, increases scrapie resistance, and maintains a high variability of PRNP. Allelic and genotypic PRNP frequencies, at the three main codons, were determined in four sheep breeds reared in central Italy (Appenninica, Bergamasca, Comisana and Sarda). Moreover homozygous ARQ sheep have been investigated to measure the frequencies of minor allelic variants. In this study the AT112RQ and ARQK176 protective allelic variants were detected. The AT112RQ allele was observed (ranging from 6.3% to 31.8%) in all the analysed breeds with the exception of Sarda sheep. The ARQK176 allele by contrast was observed only in Sarda breed (4.6%). Moreover five non synonymous polymorphisms were also found (Q101R, G127S, L141F, H143R, and H180Y). & 2015 Elsevier B.V. All rights reserved.

Keywords: Scrapie-resistance SNPs Meat breeds Dairy breeds

1. Introduction Prion (from proteinaceous infectious only) diseases, also known as TSEs (Transmissible Spongiform Encephalopathies) are a unique group of illness because they are both inheritable and infectious (Glatzel and Aguzzi, 2001). They share the pathogenetic mechanism which consists in the misfolding of the prion protein (PrPC), a cellular membrane bound protein whose function is still unclear. It can acquire pathological conformations becoming self-propagating (Prusiner, 2013). The disease-associated isoform of PrPC is termed PrPSc. The conversion of the PrPC to the aggregated form PrPSc is fundamental to the disease process and it may be that the rate of conversion is allele dependent (Hunter, 1997). TSEs in animals are known as scrapie for sheep and goats, bovine spongiform encephalopathy (BSE) for cattle, transmissible mink encephalopathy (TME) for mink, chronic wasting disease for elk, mule deer and white-tailed deer (CWD), exotic ungulate encephalopathy (EUE) for exotic ungulates and feline spongiform encephalopathy n

Corresponding author. Fax: þ39 075 5857122. E-mail address: [email protected] (E. Lasagna). 1871-1413/& 2015 Elsevier B.V. All rights reserved.

(FSE) for cats (Goldmann, 2008). The most common TSE in humans are Creutzfeldt–Jakob disease (CJD), Gerstmann–Sträussler– Scheinker syndrome (GSS) and fatal sporadic or familial insomnia (sFI, FFI) (Schneider et al., 2008; Prusiner, 2013). In the 1990s a variant of CJD (vCJD) has been discovered in relationship with Bovine Spongiform Encephalopathy (BSE) (Diack et al., 2014). In sheep single nucleotide polymorphisms (SNPs), within the open reading frame (ORF) of PRNP, influence the susceptibility to scrapie, in particular the polymorphic amino acid variations at codons 136 A/V, 154 R/H and 171 Q/R/H (Goldmann et al., 1990, Goldmann et al., 1994; Goldmann, 2008). These polymorphisms are present in 5 main allelic variants (VRQ, ARQ, AHQ, ARH and ARR) and 15 genotypes classified into 5 categories of risk according to the British National Scrapie Plan (Dawson et al., 2008). Sheep homozygous for the ARR allele (ARR/ARR) are resistant to the scrapie infection while VRQ/VRQ, ARQ/ARQ and VRQ/ARQ sheep are at high risk of developing the disease (Baylis et al., 2004). The disease is especially associated with the ARQ/ARQ genotype in sheep breeds where the VRQ allele is rare or absent. In the Mediterranean countries Italy, Greece and Spain ARQ/ARQ is a very frequent genotype detected in animals with scrapie (Nonno et al., 2003; Cosseddu et al., 2007). It is also known that not all exposed

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ARQ/ARQ (genotype based only on the three main polymorphic codons) sheep develop scrapie (Vaccari et al., 2007; Laegreid et al., 2008) and that thus the ARQ genotypes are not uniformly susceptible to scrapie. Indeed, many different and distinct variants of ARQ are known to exist in sheep (Goldmann, 2008). The genetic selection has been implemented in Europe and in United States to select sheep with genotypes that confer resistance to classical scrapie for the eradication and control of the disease (Cosseddu et al. 2007; Meydan et al. 2013). The European Union started the implementation of Genetic Selection Plans to increase the scrapie-resistant genotypes (European Commission Decision 2003/100/EC). In Belgium and in Great Britain, the use of genetic information to make genetic selection through the PrP genotyping in breeding programmes has shown to successfully increase scrapie-resistant genotypes (Dobly et al., 2013; Ortiz-Pelaez et al., 2014). Moreover in Great Britain as well as in other European countries, a statistically significant decrease in scrapie prevalence has been observed in the recent years (Hagenaars et al., 2012; Arnold and Ortiz-Pelaez, 2014; Hald and Baggesen, 2014). The variability of sheep PrP gene is not restricted to 136, 154 and 171 codons: an additional 24 polymorphic codons have been described to date, giving rise to 43 variations mainly associated with the ARQ allele (Vaccari et al., 2009). Indeed, ARQ is the most frequent allele and many authors reported ARQ-associated SNPs related to scrapie-resistance (Acutis et al., 2004; Goldmann et al., 2005; van Kaam et al., 2008; De Andreade et al., 2013; Meydan et al., 2013; González et al., 2014). Within the Sarda sheep breed, two alleles associated to classical scrapie resistance, AT137RQ and ARQK176, have been discovered (Vaccari et al., 2007; Vaccari et al., 2009; Maestrale et al., 2009; Bucalossi et al., 2011). In infected Suffolk sheep, heterozygous AM112RQ-AT112RQ showed lower attack rates and increased survival times (Chianini et al., 2013) and similarly, in a clinical study that involved the Barbado breed, heterozygous ARQ–ARK171 showed a prolonged average incubation time of 30 months before the onset of clinical signs (Greenlee et al., 2012). Moreover, the AF141RQ allele has been reported to an increased susceptibility to the atypical form of scrapie named Nor98 (Moum et al., 2005; Pongolini et al., 2009), but not with classical scrapie. Furthermore, some authors demonstrated that heterozygosity can be a protective factor by itself, as was shown in scrapie-infected murine neuroblastoma cells (Priola et al., 1994) or in infected heterozygous 171Q/R animals (Jacobs et al., 2011). The aim of this study was to evaluate PRNP polymorphisms in ARQ/ARQ animals of four main Italian sheep breeds according to their production purpose and to identify the frequency of the allelic variants. The purpose was to assess the presence and the frequency of potential protective ARQ allele variants useful for the development of breeding programmes based on genetic selection, able to increase scrapie resistance by preserving a higher variability of PRNP.


tubes added with EDTA as anticoagulant, and stored at  20 °C until analyses. The animals were sampled within the Italian Ministry of Health Research and Genotyping Program for scrapie resistance in Umbria and Marche Regions. No outbreaks were involved in the study. The differences in the number of samples and flocks per breed is proportional to the breed's census in the considered area. All the samples were collected from December 2012 to November 2013. 2.2. DNA extraction and genotyping analysis Genomic DNA was extracted by a semiautomatic extractor s (BioSprint 96, Qiagen , Hilden, Germany). Genotyping analysis of codons 136, 154, and 171 was performed following the protocol described by Vaccari et al. (2009), slightly modified: for the Allelic Discrimination Assay, 2.3 ml of genomic DNA were transferred into four different PCR mixtures (codon 136, codon 154, codon 171–1 s and codon 171–2) containing 1x TaqMan Universal PCR Master Mix, primers forward and reverse 900 nM each, and 150 nM of s TaqMan MGB probes (Table S1) to a final volume of 22 ml. Sample's genotypes were carried out with a Fast Real Time PCR Stes pOne Plus Real-time PCR System (Applied Biosystems , Foster City, CA) with the follow thermic profile: 50 °C (for 2 min), followed by 10' at 95 °C, 15'' at 95 °C for and 1' at 62 °C for 40 cycles. The results were analysed by SDS v.2.2.2 software. PrP genotypes were reconstructed on the assumption that all polymorphisms are mutually exclusive. In this study, PrP alleles are indicated with the three-letter code (e.g. ARQ or ARR) for the amino acids at positions 136, 154 and 171. Mutated alleles are indicated with the three letter code plus the additional polymorphic amino acid and its position (e.g. AT112RQ) (Vaccari et al., 2009). 2.3. Sequencing analysis The PRNP gene portion (1229 bp) where the entire CDS sequence is located, has been amplified in the homozygous ARQ samples as described by Vaccari et al. (2009) through a s s Mastercycler ep gradient S (Eppendorf , Hamburg, Germany). PCR s fragments were purified with QIAquick PCR purification kit s (Qiagen , Hilden, Germany) according to manufacturer's recommendations. Purified amplicons have been sequenced for both the strands. Sequencing reaction was carried out with primers T3 (5′-TTTACGTGGGCATTTGATGC-3′) and T4 (5′-GGCTGCAGGTAGACACTCC-3′) (Vaccari et al., 2009) using Big Dye Terminator Cycle s sequencing Kit v1.1 (Applied Biosystems , Foster City, CA, USA) and s detected with ABI PRISM 3100 apparatus (Applied Biosystems , Foster City, CA, USA). Individual chromatograms were read and sequences were aligned to the Ovis sp. gene for prion protein PrP, complete cds (Accession number, GenBank D38179.1) with ClustalW tool of BioEdit 7.0.9 software (Hall, 1999). 2.4. Data analysis

2. Materials and methods 2.1. Samples collection A total of 647 whole blood samples, only female (with a minimum age of 18 months), belonging to the meat breeds Appenninica (n ¼169, 12 flocks) and Bergamasca (n ¼100, 4 flocks) and to the dairy breeds Comisana (n ¼100, 5 flock) and Sarda (n ¼278, 9 flocks) were collected. For each breed the 30% of the flocks in Umbria and Marche region were randomly chosen. Inside each flock 10% of the animals were randomly chosen. Blood sams ples were collected from each animal with Vacutainer system, in

The allelic and genotypic frequencies, separately per breed, were estimated. Moreover, to observe if the physiological pattern of the sheep should be related to susceptibility/resistance, the differences between the productive purposes (milk or meat) were also evaluated by aggregating the breeds (Appenninica and Bergamasca vs. Comisana and Sarda, respectively). The statistical significance of differences in allelic and genotypic frequencies between the breeds was evaluated by the Chi-square test implemented in the Microsoft Excel software. Moreover, deviation from Hardy–Weinberg equilibrium in each breed was evaluated with the Chi-square test using R software (R Development Core Team, 2013).


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3. Results and discussion

Table 2 Genotypic frequencies (%).

ARQ was the predominant allele, followed by the ARR; ARH, AHQ, and VRQ alleles were detected at low frequencies (Table 1). Almost all the allelic frequency values were significantly different among breeds (Pr 0.005). The Bergamasca sheep presented the highest frequency of the ARQ compared to all the other breeds studied. Sarda sheep had a higher percentage of the resistant ARR allele than the other breeds. Among the dairy sheep breeds, the Comisana breed had a greatest ARQ allele frequency. In general, the ARQ frequencies were similar to those obtained by Pongolini et al. (2009) in the same breeds and in Massese breed (not investigated in this paper). However, the ARR frequency of 6.9% reported by Pongolini et al. (2009) for Bergamasca was much lower, perhaps due to different husbandry areas of the sheep population analysed. Both the ARQ and ARR alleles in the Sarda breed were similar to that reported by Vaccari et al. (2009). The frequencies of the alleles observed in this study are in agreement with other researches. ARQ was the predominant allele in Canadian sheep (Harrington et al., 2010), in Chinese Hu sheep (Guan et al., 2011), in Finnish sheep (Hautaniemi et al., 2012) and also in Tunisian sheep (Kdidi et al., 2014). For the past decade, the more common allele in Belgium (79.3%) and in Great Britain (52.3%) has been ARR (Dobly et al., 2013; Ortiz-Pelaez et al., 2014); these values are quite higher than the ARR frequencies observed in this study, certain due to the effect of different European Genetic Selection Plans. In Table 2, the highest three genotypic frequencies were ARR/ ARR, ARR/ARQ, ARQ/ARQ; the observed values were comparable to that reported by Vaccari et al. (2009) in the Sarda breed and to the ones reported by Pongolini et al. (2009) in the Appeninica, Comisana and Sarda breeds. The ARR/ARQ genotype was the most frequent also in British sheep population (29.5%), followed by ARR/ ARR (28.6%), ARR/AHQ (10.9%), ARQ/ARQ (9.8%) and AHQ/ARQ (7.2%) (Ortiz-Pelaez et al., 2014). The frequencies of the ARR/ARQ, ARQ/ARQ, ARR/ARR genotypes are similar to those reported by Harrington et al. (2010) in Canadian sheep and by Guan et al. (2011) in Chinese Hu sheep. In contrast, the ARQ/ARQ is the most frequent genotype registered in many countries, followed by ARR/ ARQ and ARR/ARR: a similar trend was shown in sheep breeds of northwestern China (Zhao et al., 2012) and in Finnish sheep (Hautaniemi et al., 2012). A Belgian sheep population resulted to have ARR/ARR as dominant genotype (Dobly et al., 2013). Based on Fisher's exact test, each breed was in Hardy–Weinberg equilibrium for all genotypes and no deviation was detected (P 40.05). Furthermore the genotypic frequencies estimated from allelic frequencies are very close to that directly computed on the breeds; the small observed differences could be due to the sampling features or to the selection favouring the resistant genotypes carried out in the last years. A total of 146 ARQ/ARQ animals located in the restricted area of the Umbria and Marche regions were found: 64 in Appenninica, 76 in Bergamasca, 44 in Comisana and 108 in Sarda. In this work, seven non-synonymous (Q101R, M112T, G127S, L141F, H143R, Table 1 Allelic frequencies (%). Alleles APN (N ¼ 338)

BGS (N ¼ 200)

CMS (N ¼ 200)

SRD (N ¼ 556)



30.5 0 4.0 64.5 1.0

36.0 2.5 8.0 47.0 6.5

47.7 6.1 0.7 44.2 1.3

r 0.05 r0.005 r0.005 r0.005 r0.005

42.3 1.5 8.0 43.2 5.0

Breeds: APN, Appenninica; BGS, Bergamasca; CMS, Comisana; SRD, Sarda N: total number of alleles


APN (n¼169)

ARR/ARR 19.5 ARR/AHQ 2.4 ARR/ARH 5.9 ARR/ARQ 34.3 AHQ/AHQ 0.0 ARQ/AHQ 0.6 ARH/AHQ 0.0 ARH/ARH 0.6 ARQ/ARH 7.7 ARQ/ARQ 18.9 ARQ/VRQ 5.9 ARR/VRQ 3.0 AHQ/VRQ 0.0 ARH/VRQ 1.2 VRQ/VRQ 0.0 HWE (P-value) 0.718*

BGS (n ¼100)

CMS (n ¼100)

SRD (n¼ 278)


7.0 0.0 1.0 45.0 0.0 0.0 0.0 0.0 7.0 38.0 1.0 1.0 0.0 0.0 0.0 0.482*

13.0 2.0 6.0 32.0 0.0 3.0 0.0 0.0 9.0 22.0 6.0 6.0 0.0 1.0 0.0 0.994*

24.5 4.3 0.0 40.6 0.7 6.5 0.0 0.0 1.4 19.4 1.1 1.4 0.0 0.0 0.0 0.336*

r 0.01 NS r 0.005 NS NS r 0.005 ND NS r 0.005 r 0.005 r 0.01 NS ND NS ND

Breeds: APN, Appenninica; BGS, Bergamasca; CMS, Comisana; SRD, Sarda n: total of animals. *

P4 0.05; NS: no statistically significant; ND: not determinated

Table 3 ARQ/ARQ sheep breeds frequencies (%) of PrP polymorphisms. Codon

101* 112* 127* 141* 143* 176* 180* 231* 237*

(wt) (mut) (wt) (mut) (wt) (mut) (wt) (mut) (wt) (mut) (wt) (mut) (wt) (mut) (wt) (mut) (wt) (mut)



92.2 7.8 93.8 6.3 100.0 0.0 92.2 7.8 100.0 0.0 100.0 0.0 98.5 1.6 87.5 12.5 87.5 12.5

BGS (N¼ 76)

CMS (N ¼44)

SRD (N¼ 108)

100.0 0.0 85.5 14.5 100.0 0.0 100.0 0.0 96.1 3.9 100.0 0.0 100.0 0.0 85.5 14.5 88.2 11.8

100.0 0.0 68.2 31.8 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 100.0 0.0 88.6 11.4 88.6 11.4

100.0 0.0 100.0 0.0 98.1 1.9 99.1 0.9 100.0 0.0 95.4 4.6 100.0 0.0 91.7 8.3 91.7 8.3

Breeds: APN. Appenninica; BGS. Bergamasca; CMS. Comisana; SRD. Sarda wt: wild type; mut: mutation N: total number of animals * The differences between genotypes are significant at χ2 test in all the codons (P r0.05)

N176K, and H180Y) as well as two synonymous polymorphisms at codons 231 and 237 were observed and their frequencies have been calculated for each sheep breed (Table 3). Alleles reconstruction has been made with the notion that when additional polymorphisms are found those are computed to the ARQ (the ancestral) allele. For Biellese breed, some polymorphisms within ARQ have been already reported at codons 141, 143 and 180 (Acutis et al., 2004). The ARQ/ARQ animals of the Sarda breed showed polymorphisms at codons 112, 127, 137, 141, 142, 143 and 176 (Vaccari et al., 2007; Vaccari et al., 2009; Maestrale et al., 2009; Bucalossi et al., 2011). Zhao et al. (2012) detected some of these ARQ polymorphisms, in northwestern China sheep, specifically at codons 127, 141 and 143, while 10 polymorphisms (21, 101, 112, 127, 138, 141, 143, 146, 153 and 189) were identified in 16 Chinese local sheep breeds (Lan et al., 2014); nine SNPs were detected in Chinese Hu sheep (Guan et al., 2011). In 18 Turkish native sheep breeds were found 22

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aminoacid polymorphisms and three silent mutations in codons 101, 112, 127 and 143 (Meydan et al., 2013). The SNPs AF141RQ and AR143RQ were also found in Finnish sheep breeds (Hautaniemi et al., 2012). The allelic variant AR101RQ was found in the Appenninica breed but it was not detected in any of the other three breeds. This amino acid substitution has been shown with a frequency ranging from 0.7 to 10.9% in four Spanish sheep breeds (Acín et al., 2004); the same results were observed in a Chinese sheep breed (Guan et al., 2011; Lan et al., 2014) and in a Turkish sheep breed (Meydan et al., 2013). The high frequency of the AT112RQ detected in Comisana has to be considered; the same was not observed in Sarda breed and this result is in contrast with that obtained by Vaccari et al., (2009) maybe because of the different husbandry region areas. The AT112RQ allele has been reported in other sheep populations in the world such as Spanish, Chinese and Turkish (Acín et al., 2004; Guan et al., 2011; Lan et al., 2014; Meydan et al., 2013). Hautaniemi et al. (2012), found an association between AT112RQ and a partial resistance with natural scrapie in Japanese Suffolk and Corriedale sheep; Saunders et al. (2009) in Suffolk sheep, supposed a BSE resistance in British sheep due to AT112RQ. In our study, the Sarda breed is the only one to have the AS127RQ allele as reported by Guan et al. (2011), Lan et al. (2014) and Zhao et al. (2012) in different Chinese sheep populations (Table 3). Vaccari et al. (2009), in Sarda sheep breed did not detect AS127RQ but V127ARQ allele was observed, probably because of differing regional husbandry practices for sheep populations. AF141RQ allele, responsible of susceptibility to atypical scrapie, was not detected in the Bergamasca and Comisana; however in other Italian studies it was detected in Appenninica, Bergamasca, Biellese and Sarda breeds so, again, perhaps these results were due to differing regional sheep rearing (Acutis et al., 2004; Maestrale et al., 2009; Pongolini et al., 2009; Vaccari et al., 2009). AF141RQ allele was also found in Spanish sheep (Acín et al., 2004), in Finnish sheep (Hautaniemi et al., 2012) and in Chinese local sheep breeds (Lan et al., 2014). In the Comisana breed only the AL141RQ allele was present according to Pongolini et al. (2009): in our study the Bergamasca sheep showed for the same allele a different frequency from that reported in this previous work, the reason might be explained by genetic variability associated with geographic area. As reported in Table 3, only Bergamasca sheep presented the AR143RQ allele in a low frequency; this mutation has been also detected in Spanish sheep breeds (0–16.3%) by Acín et al. (2004), in Finnish Aland breed (1.0%) (Hautaniemi et al., 2012), and in Italian Biellese breed (0.2%) (Acutis et al., 2004). Data analysis confirmed the presence of ARQK176 exclusively in the Sarda sheep breed according with Bucalossi et al. (2011), Maestrale et al. (2009) and Vaccari et al. (2009). In some Spanish breeds ARQK176 has a frequency ranging from 0.8 to 5.4% (Acín et al., 2004). In this study we did not find any AT137RQ mutation associated with resistance and found in Sarda breeds from other regions (Sardegna, Toscana, Lazio). The Appenninica breed showed a low frequency of ARQY180 polymorphism similar to the Biellese breed described by Acutis et al. (2004). The mutations detected at the codon 127 and 180 seem not to have an effect on susceptibility/resistance to scrapie. As reported by Acín et al. (2004), AR101ARQ and AR143QR alleles in scrapieaffected animals were detected. The synonymous polymorphisms at codon 231 and 237 were observed in all the analysed breeds. 4. Conclusion In Europe selection programs to increase the scrapie resistance of ovine population by increasing the ARR frequencies are giving


the first results. Indeed data from several countries showed a statistically significant decrease in scrapie prevalence. Although not yet integrated in the European Commission guidelines some authors suggested the use of additional resistant alleles in order to preserve PRNP variability. This approach, although very interesting, should be supported by the implementation of, not yet available, specific methods for the analysis of the genotypes at additional codons. This paper highlights that SNPs investigated are not ubiquitously present in all the four Italian sheep breeds. We detected seven non-synonymous (Q101R, M112T, G127S, L141F, H143R, N176K, and H180Y) and two synonymous polymorphisms at codons 231 and 237. Some PRNP polymorphisms found at codons 101, 112, 141 and 176 could have a protective role to scrapie. The allelic variant N176K shows a low frequency of this protective allele in the Sarda breed so, selection will not be simple. However, the high frequency of the AT112RQ found in Comisana sheep could be used in future planning of genetic scrapie-resistance selection. Scrapie infection studies and case reports are needed to understand the role of polymorphisms on the resistance/susceptibility to scrapie disease. It is also necessary to know whether these mutations have effect either alone or in association with others. The results obtained about the allelic variant frequencies could offer the opportunity to develop a genetic breeding programme aimed at increasing resistance in sheep populations preserving PRNP variability, with a subsequent decrease in the number of animals culled, particularly during scrapie outbreaks, thus significantly reducing the costs involved for cull compensation claims.

Conflict of interest The authors declare that there are no conflicts of interest.

Acknowledgements The study was supported by Italian Ministry of Health grant IZSUM 08/2011 RC (Valutazione di Nuovi Alleli Protettivi per la Scrapie AT137RQ/ARQK176 nelle Razze Ovine presenti nel Territorio Umbro Marchigiano). A special thanks to Dr. S. Ciancaleoni (University of Perugia, Italy) for the support in the statistical analysis. The authors wish to thank the two anonymous referees for their valuable comments to the manuscript and their constructive suggestions.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at

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