PRNP polymorphisms in Greek goats affected with natural scrapie Evridiki Boukouvala1*, Efstathios Katharopoulos1, Paula Stewart2, Vayia Palaska3, Nektarios Giadinis4, Georgios Arsenos4, Loukia V. Ekateriniadou1, Wilfred Goldmann2, Jan P.M. Langeveld5 1Veterinary Research Institute of Thessaloniki; Thessaloniki, Greece; 2 The Roslin Institute, R(D)SVS University of Edinburgh, Midlothian, UK; 3National Reference Laboratory for TSEs, Larissa, Greece; 4Aristotle Universtity of Thessaloniki, Thessaloniki, Greece; 5Central Veterinary Institute of Wageningen UR (CVI-WUR), Lelystad, The Netherlands Introduction. As a part of an EU EMIDA ERA-NET project entitled “Towards breeding of goats for genetically determined TSE resistance” we analyzed 296 brain tissue samples from scrapie affected Greek goats for PRNP gene variability and disease association. Here we focused on five polymorphisms of caprine PRNP: T110P, N146S, N146D, R211Q and Q222K. With the exception of codon 110 all have previously been associated with scrapie resistance and are rarely found in scrapie animals (Acutis et al., 2006; Barillet et al., 2009; Papasavva Stylianou et al., 2011; Fragiadaki et al. 2011; Corbiere et al., 2013). This study revealed an unexpectedly high frequency of the 222K allele in the scrapie positive samples. Material and Methods. In total, 296 brain tissue samples from naturally affected goats were analyzed from 71 herds. Genomic DNA was extracted using the PureLink Genomic DNA Mini Kit (Life Sciences). The PRNP coding region was amplified in specific DNA fragments for each polymorphism and genotyped by real time PCR. The list of oligonucleotides can be send on request (boukouvala@vri.gr). In a selected number of animals direct sequencing of the full coding region and the basic PRNP gene promoter were also performed (Goldmann et al., 2011). The SPRN gene encoding Shadoo protein was genotyped at position 602 for animals with genotype 222QK (Peletto et al., 2012). Results. All goats were PRNP genotype 146NN equivalent to wild type PrP. The polymorphism T110P was detected in 2 animals (0.67%) from 2 herds of the highest incidence’ region while the polymorphism R211Q was detected in 11 animals (3.7%) from 4 herds. Surprisingly, the polymorphism Q222K was detected in 13 samples from 5 herds with a total population of 1138 animals (Table 1). When sorted by region, 222QK genotypes were found at a frequency as high as 37.5% in the scrapie goats (Table 2). So far only 40 negative controls could be collected, of those the majority were 110TT, 146NN, 211RR and 222QQ (Table 3); moreover genotyping of 28 healthy herds with 1060 animals revealed K222 carrier frequencies from 0% in 11 herds to 2-20% in the other 17 herds (mean 5%). The positive 222QK goats were analysed in more detail to exclude the possibility that their unexpectedly high occurrence in scrapie-affected goats was linked to an unusual PRNP gene haplotype or a known scrapie-associated polymorphism in the SPRN gene (Peletto et al 2012) (Table 4). The results so far do not indicate a shared unique haplotype in these samples, a comparison with negative controls from these regions will be done when samples become available. codon genotype No. of goats % No herds 110 TP 2 0.67 2/71 146 NN 296 100 71/71 211 RQ 11 3.7 4/71 222 QK 13 4.38 5/71 Total positive goats   Region Q/K goats total positive % Larisa 9 24 37.5 Pella 2 29 6.90 Ioannina 14 14.28 Total 13 67 100 Table1. PRNP genotype frequencies in positive brain samples Table 2. Distribution by regions of goats with 222QK genotype. codon polymorphism No % 110 TP 5 12.5 146 NS 2 5.0 211 RR 40 100 222 QK Total negative samples   Conclusion. The absence of the 146S polymorphism in scrapie positive Greek goats (3-9% in Greek goats free of scrapie disease, Fragkiadaki et al, 2011 and unpublished) supports the hypothesis that it is associated with resistance. The observation of scrapie-positive K222 carriers in the three different geographical areas and in 5 out of 71 infected flocks was surprising based on previous studies that had shown strong association of K222 with resistance (Corbiere et al., 2013). Our study appears to indicate a higher incidence of scrapie in 222K carriers than reported for French goats. There was no evidence from the PRNP gene sequences that the K222 allele in these Greek goats contains additional mutations associated with susceptibility nor that there is a particular SPRN allele. The fact that all samples derived from mixed flocks with a high prevalence of sheep scrapie and the presence of the disease for more than two years have to be considered as factors. We propose that other factors such as a particularly strong infection pressure in these herds or a different scrapie strain may also contribute to this situation. A future case-control study is necessary to ascertain the factors of the unique scrapie infection circulating in Greek infection areas. Table 3. PRNP genotype frequencies in scrapie-negative goats SPRN 602_606insCTCCC * No % wildtype / insertion 4 30.8 wildtype / wildtype 9 69.2 insertion / insertion total 13   Table 4. SPRN InDel genotypes according to Pelletto et al (2012) for scrapie goats with PRNP genotype 222QK References Papasavva-Stylianou P. et al (2011) PrP gene polymorphisms in Cyprus goats and their association with resistance or susceptibility to natural scrapie.”Vet J, 187,45–250. Acutis PL, et al (2006) Identification of prion protein gene polymorphisms in goats fromItalian scrapie outbreaks.” J Gen Virol , 87, 1029–1033. Barillet F, et al (2009) Identification of seven haplotypes of the caprine PrP gene at codons 127, 142, 154, 211, 222 and 240 in French Alpine and Saanen breeds and their association with classical scrapie.”, J Gen Virol , 90, 769–776. Corbiere F, (2013) PrP-associated resistance to scrapie in five highly infected goat herds. J Gen Virol 94, 241–245 Fragkiadaki EG, (2011) PRNP genetic variability and molecular typing of natural goat scrapie isolates in a high number of infected flocks. Vet Res, 2011, 42:104 Goldmann et al. (2011) Caprine prion gene polymorphisms are associated with decreased scrapie susceptibility in goat herds in the United Kingdom. Vet Res 42, 110 Peletto et al (2012) Association of an indel polymorphism in the 3’UTR of the caprine SPRN gene with scrapie positivity in the central nervous system. J Gen Virol 93, 1620-1623