Curcio_imp:ok 20-06-2016 11:34 Pagina 259 L. Curcio et al. Large Animal Review 2015; 21: 259-262 259 A study on PrP genotype frequencies in meat and dairy sheep breeds reared l in Umbria and Marche regions L. CURCIO1,2, C. MARESCA1, C. SEBASTIANI1, M. CIULLO1, A. DETTORI1, E. LASAGNA2, M. BIAGETTI1 1 Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche - Area Ricerca e Sviluppo Via G. Salvemini, 1 - 06126 Perugia (Italy) 2 Dipartimento di Scienze Agrarie, Alimentari e Ambientali - Università degli Studi di Perugia Borgo XX Giugno, 74 - 06121 Perugia (Italy) SUMMARY Introduction - In sheep, susceptibility/resistance to scrapie is mainly associated to the polymorphisms of PRNP gene at co- dons 136, 154 and 171. Five alleles are usually present (ARR, ARQ, ARH, AHQ, VRQ) and they have a crucial role in terms of incubation period and/or susceptibility to scrapie. Aim - In this study we analysed PrP genotypes obtained from different sheep breeds reared in central Italy (Umbria and Mar- che regions) during last seven years in order to gain knowledge of the allelic and genotypic frequencies and of the correlation between genotype, breed and productive purpose. Materials and methods - A total of 21152 animals belonging to half-breeds and 8 purebreeds (Merinizzata, Bergamasca, Me- rinos, Fabrianese, Appenninica, Sarda, Massese and Comisana) were genotyped by Real time PCR. Results and discussion - The ARQ and ARR were predominant alleles. The frequencies of the ARR/ARQ, ARQ/ARQ, ARR/ARR genotypes overlay with other studies. Considering the correlation between susceptible genotypes and productive purpose, meat breeds had a higher number of susceptible genotypes than dairy and dual purpose breeds. Conclusion - The results showed that Sarda, Comisana, Massese and Merinos breeds have a lower probability to be affected by scrapie than the other breeds analysed and so they could be classifiable as breeds with a good level of genetic resistance. As re- gards to the other breeds, they have a medium to high level of genetic susceptibility to scrapie. KEY WORDS Scrapie susceptibility, scrapie resistance, PRNP. INTRODUCTION genotypes that show resistance to classical scrapie2. PrP ge- notyping was adopted for planning selective breeding pro- Scrapie belongs to the group of Transmissible Spongiform grammes and for the management of disease outbreaks in Encephalopathies (TSEs) and affects sheep and goats. These order to decrease the incidence of scrapie. European Com- diseases are characterized by the accumulation in the central mission Decision 100/2003/EC implemented a breeding pro- nervous system of an abnormal conformational form (PrPSc) gram that promoted positive selection of the ARR allele and of a natural host-encoded protein, the prion protein (PrPC)1. the gradual elimination of the VRQ allele7. The application Scrapie is strongly associated to the polymorphisms of PRNP of these measures is able to reduce scrapie in Europe, but the gene at codons 136, 154 and 171, that determine the resistan- risk that a new outbreak of scrapie in sheep may arise it is ce/susceptibility of sheep2. These polymorphisms are present difficult to predict5. in 5 main different alleles: ARR, ARQ, ARH, AHQ and VRQ. The purpose of this paper was to evaluate the allelic and ge- In addition, more rarely, ARK, TRQ and ALQ alleles have notypic frequencies at PRNP gene in Merinizzata, Bergama- been described at the same codons. In particular, ARK (iden- sca, Fabrianese, Comisana, Appenninica, Sarda, Massese and tifying 171-lysine) has been detected with a certain frequency Merinos breeds, as well as in half-breed animals, in order to in Bergamasca, Appenninica and Biellese Italian breeds3,4. assess the current level of resistance to classical scrapie. Besi- Ovine homozygous for the ARR allele (ARR/ARR) are the des the correlation between resistance/susceptibility and the most resistant to classical scrapie, while VRQ/VRQ, productive purposes of the studied breeds was investigated. ARQ/ARQ and VRQ/ARQ are the most susceptible genoty- pes5,6. The demonstrated association between PrP genotype and scrapie susceptibility led a number of countries to adopt MATERIALS AND METHODS genetic selection programs to increase the frequency of the Animal sampling Animals belonging to half-breed, seven Italian breeds (Meri- nizzata, Bergamasca, Fabrianese, Appenninica, Sarda, Masse- Autore per la corrispondenza: se and Comisana) and Merinos breed of Spanish origin but Ludovica Curcio ([email protected]). raised in Italy, were examined. During the period from 2006 Curcio_imp:ok 20-06-2016 11:34 Pagina 260 260 A study on PrP genotype frequencies in meat and dairy sheep breeds reared in Umbria and Marche regions Table 1 - Number of sheep analyzed for each breeds, listed ac- cording to their productive purpose (meat, milk, dual). Meat Milk Dual MRZ 4808 SRD 5775 MTT 3700 BGS 1326 MSS 882 MRN 1223 CMS 85 FBN 172 MTT 1831 APN 150 MTT 1200 TOTAL 8879 8573 3700 MRZ: Merinizzata; BGS: Bergamasca; MRN: Merinos; FBN: Fabrianese; APN: Appenninica; MTT: Half-breed; SRD: Sarda; MSS: Massese; CMS: Comisana. Figure 1 - Allelic frequencies in the studied breeds reared in Um- bria and Marche regions from 2006 to 2012. to 2012, a total of 21152 whole peripheral blood samples col- lected in EDTA tubes were genotyped from sheep flocks co- ming from Central Italy (Table 1). The animals were separa- RESULTS ted per productive purpose on the basis of the breed or of the farmer informations for half-breed animals. These breeds are The allelic and genotypic frequencies of PRNP gene, analy- commonly raised in this geographic area (Umbria and Mar- sed by Real Time PCR to obtain quickly the results of alto- che regions) for meat or milk production or dual purpose. gether 21152 purebreed and half-breed sheep considering the codons 136, 154, and 171 are shown in Figures 1 and 2. Genotyping analysis In the studied breeds, the genotypic frequencies were descri- by Real Time PCR bed by a histogram highlighting ARR/ARQ, ARQ/ARQ and DNA from samples was extracted with the High Pure PCR ARR/ARR as the main genotypes. A second histogram was Template Preparation Kit (Roche Applied Science, created for the allelic frequencies and the ARQ allele resulted Mannheim, Germany) according to manufacturer’s recom- the predominant one, followed by ARR allele (Fig. 1). In Ta- mendations and analysed by Real Time PCR, using an allelic ble 1, each breed was included in a specific group on the ba- discrimination protocol able to detect A (Alanine) and V (Va- sis of productive purpose. line) at codon 136, R (Arginine) and H (Histidine) at codon Considering the susceptible genotypes and sheep productive 154, Q (Glutamine), R (Arginine) and H (Histidine) at codon purpose, the meat breeds showed 53% of the susceptible ge- 1718. The PCR reaction mix contained 1X TaqMan® Univer- notypes while in the dairy breeds and dual purpose were sal PCR Master Mix UNG, forward and reverse primers 900 34% and 47%, respectively (Table 2). Dairy breeds showed nM each, variable concentrations of TaqMan® MGB probes the highest percentage of the resistant genotypes (66%) fol- to a final volume of 22 µl8. For the Allelic Discrimination As- lowed by dual purpose and meat breeds (53% and 47%, re- say, 2.3 µl of genomic DNA were transferred into four diffe- spectively) (Table 3). Half-breed, Merinizzata, Fabrianese rent PCR mixtures (codon 136, codon 154, codon 171.1 and and Bergamasca breeds showed over 40% of susceptible ge- codon 171.2). The thermal profile consisting of a step at 50°C notypes compared to the other breeds (Table 4). for 2’, followed by a step at 95°C for 10’, and 40 cycles at 95°C Odds Ratio (OR) was calculated for half-breed and for the for 15’’ and at 62°C for 1’. Genotyping was carried out on a eight studied breeds: Sarda, Merinizzata, Merinos, Bergama- Real Time PCR ABI PRISM 7900HT and Stepone Plus (Ap- sca, Massese, Appenninica, Fabrianese, Comisana (Table 5). plied Biosystems®, Foster City, CA). This method was valida- OR values of the half-breed were 1.10 with P-value = 0.0020 ted/accredited according to UNI EN ISO 17025/2005. (C. I. 95% 1.03-1.07). OR values showed that Merinizzata, Bergamasca and Fabria- Statistical analysis nese had, respectively, 2, 1.5 and 2.5 fold, higher chance to Statistic descriptive analysis were performed to measure the have susceptible genotypes than Sarda (P-value < 0.05). OR allelic and genotypic frequencies, the distribution of suscep- values for Massese and Comisana breeds differences (OR <1) tible/resistant genotypes by productive purpose and by were statistically significant. OR value was not statistically si- breed. Odds Ratio (OR) was used as measure of association gnificant in Merinos, and Appenninica breeds (Table 5). between genotypes and breed of the sheep and to determine whether the breed is a risk factor for having a susceptible or resistant genotype for scrapie and to compare the magnitu- DISCUSSION de of risk. Sarda breed started from a very good percentage of resistant animals and was used as baseline. The 95% con- The effect on the genotype frequencies of the selective bree- fidence interval (C. I.) was used to estimate the precision of ding could not be considered; in fact, the years data were the OR. Statistically significant data were considered for P- gathered all together, thus the obtained frequencies could be value ≤0.05 calculated by two-tailed Chi-square test. Statisti- a bit divergent from the real present frequencies. However, cal analysis was performed with Stata software, version 11.1 the overall frequency, reported in the histogram (Fig. 1, 2), (Copyright 2009 Stata Corp LP Stata Corp). was similar to those obtained by Pongolini et al.3, and by Vac- Curcio_imp:ok 20-06-2016 11:34 Pagina 261 L.