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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 and 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 () 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, , , Sarda, Massese and ) 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 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. Curcio et al. Large Animal Review 2015; 21: 259-262 261

Figure 2 - Genotypic frequencies in the studied breeds reared in Umbria and Marche regions from 2006 to 2012.

Table 2 - Distribution of susceptible genotypes, separately per productive purpose.

Productive purpose Susceptible genotypes Meat Milk Dual n. 8879 % n. 8573 % n. 3700 % ARQ/ARQ 2881 32.5 2455 28.6 1311 35.4 ARQ/AHQ 399 4.5 280 3.3 65 1.8 ARQ/VRQ 356 4 52 0.6 149 4 ARQ/ARH 321 3.6 27 0.3 65 1.8 AHQ/ARH 89 1 – – 12 0.3 VRQ/ARQ 17 0.2 1 0.01 13 0.4 AHQ/AHQ 8 0.1 18 0.2 2 0.05 ARH/ARH 24 0.2 – – 2 0.05 AHQ/VRQ 18 0.2 3 0.03 3 0.08 ARH/VRQ 16 0.2 – – 3 0.08 ARR/VRQ 596 6.7 46 0.5 120 3.2 Susceptible genotypes 4725 53% 2882 34% 1745 47% per productive purpose

Table 3 - Distribution of resistant genotypes, separately per productive purpose.

Productive purpose Resistant genotypes Meat Milk Dual n. 8879 % n. 8573 % n. 3700 % ARR/ARQ 2484 28 3817 44.5 1366 36.9 ARR/ARR 1294 14.6 1578 18.4 502 13.6 ARR/AHQ 173 1.9 265 3.1 52 1.4 ARR/ARH 203 2.3 30 0.4 35 0.9 Resistant genotypes 4154 47% 5690 66% 1955 53% per productive purpose Curcio_imp:ok 20-06-2016 11:34 Pagina 262

262 A study on PrP genotype frequencies in meat and dairy sheep breeds reared in Umbria and Marche regions

Table 4 - Distribution of resistant and susceptible genotypes, se- 4. In Massese and Comisana, both dairy breed, OR data ha- parately per breed. ve showed a negative statistical association: these breeds Susceptible Resistant could have a good level of resistant genotypes breeds com- Breed N. % N. % pared to Sarda. SRD 1831 33 3644 67 MRZ 2460 51 2348 49 CONCLUSION MRN 431 35 792 65 BGS 562 42 764 58 It was evident that meat breeds showed higher susceptible genotypic frequencies than dairy breeds. Probably there has MSS 171 19 711 81 been a major attention on dairy sheep because, almost in APN 54 36 96 64 Italy, milk production was the most important production FBN 98 57 74 43 respect to meat breed. The dairy sheep farms were highly de- CMS 15 18 70 82 veloped in many countries, especially in the Mediterranean area and the dairy ewes are usually intensively managed for MTT 2698 42 3733 58 high milk production. Moreover, as milk sheep was associa- SRD: Sarda; MRZ: Merinizzata; MRN: Merinos; BGS: Bergamasca; MSS: ted with important economic interests, they have been rigo- Massese; APN: Appenninica; FBN: Fabrianese; CMS: Comisana; MTT: Half-breed. rously selected through the PrP genotyping in order to im- prove the scrapie resistance of the ovine flocks.

Table 5 - Statistical analysis performed by Odds ratio (OR) to de- ACKNOWLEDGEMENTS termine the association between genotypes and breed.

Breed OR C.I. 95% P-value The study was supported by Italian Ministry of Health grant IZSUM 08/2011 RC (Valutazione di nuovi alleli protettivi Sarda 1 per la Scrapie AT137RQ/ARQK176 nelle razze ovine presen- Massese 0.5 0.4-0.6 0.0000 ti nel territorio umbro marchigiano). Comisana 0.4 0.2-0.7 0.0022 Merinizzata 2 1.9-2.2 0.0000 Merinos 1.0 0.9-1.2 0.2293 References

Bergamasca 1.5 1.3-1.6 0.0000 1. Prusiner, S.B., 2013. Biology and genetics of prions causing neurodege- neration. Annu. Rev. Genet. 47:601-623. Appenninica 1.1 0.8-1.6 0.5127 2. Goldmann, W., Baylis, M., Chicota, C., Stevenson, E., Hunter, N., 2005. Fabrianese 2.6 1.9-3.6 0.0000 Frequencies of Prp gene haplotypes in British sheep flocks and the im- plications for breeding programmes. J. Appl. Microbiol. 98:1294-1302. Merinizzata, Bergamasca and Fabrianese breeds, in black bold font, could 3. Pongolini, S., Bergamini, F., Iori, A., Migliore, S., Corradi, A., Bassi, S. be considered with a low level of genetic resistance. Massese and Comi- 2009. Prion protein genotypes of italian sheep breeds with lysine-171 sana breeds, in red bold font, could be classifiable with a good level of ge- and phenylalanine-141 detection. Vet Microb. 137:18-23. netic resistance. 4. Acutis, P.L., Sbaiz, L., Verburg, F., Riina, M.V., Ru, G., Moda, G., Cara- ≤ P-value: statistically significant (P 0.05). melli, M., Bossers, A., 2004. Low frequency of the scrapie resistance as- C.I.: Confidence Interval. sociated allele and presence of lysine-171 allele of the prion protein ge- ne in Italian Biellese ovine breed. J. Gen. Virol. 85:3165-3172. 5. Dawson, M., Moore, R.C., Bishop, S.C., 2008. Progress and limits of PrP gene selection policy. Vet. Res. 39(4):25. 8 6. Goldmann, W., 2008. PrP Genetics in ruminant transmissible spon- cari et al. , confirming our results. In Belgium and in Great giform encephalopathies. Vet. Res. 39:30. Britain, the ARR values (79.3% and 52.3%, respectively) we- 7. COMMISSION DECISION of laying down minimum requirements for re quite higher than the ARR frequencies observed in this the establishment of breeding programmes for resistance to transmissi- ble spongiform encephalopathies in sheep. O J L 41, 13.2.2003 p. 41-43. study, because the use of genetic selection through the PrP 8. Vaccari, G., Scavia, G., Sala, M., Cosseddu, G., Chiappini, B., Conte, M., genotyping in breeding programmes has shown to succes- Esposito, E., Lorenzetti, R., Perfetti, G., Marconi, P., Scholl, F., Barbaro, sfully increase scrapie-resistant genotypes9,10. K., Bella, A., Nonno, R., Agrimi, U., 2009. Protective effect of the Analysing data of susceptible and resistant genotypes, in AT137RQ and ARQK176 PrP allele against classical scrapie in Sarda breed sheep. Vet. Res. 40:19. Tables 2 and 3, we confirmed that milk breeds had a better 9. Dobly, A,. van der Heyden, S., Roels, S., 2013. Trends in genotype fre- level of genetic resistance than dual purpose and meat quency resulting from breeding for resistance to classical scrapie in Bel- breeds. OR data have showed that half-breed as well as Me- gium (2006-2011). J. Vet. Sci. 14:45-51. 10. Ortiz-Pelaez, A., Thompson, C.E., Dawson, M., 2014. The impact of the rinizzata and Fabrianese breed (Table 5) had a low level of National Scrapie Plan on the PRNP genotype distribution of the Briti- genetic resistance according to the results reported in Table sh national flock, 2002-2012. Vet. Rec. 174:530.