Slov Vet Res 2004; 41 (2): 83-88 UDC 619:616.831:575.1:636.3
Original Research Paper
SCRAPIE-SUSCEPTIBILITY-LINKED POLYMORPHISMS OF THE PRION PROTEIN GENE IN ISTRIAN PRAMENKA SHEEP
Jelka Zabavnik 1*, Marko Cotman 2, Milan Pogačnik 3, Polona Juntes 3
Addresses of authors: 1 Institute of Anatomy, Histology and Embryology, 2 Clinic for the Health Care of Pigs, 3 Institute of Pathology, Administrative and Forensic Veterinary Medicine, Veterinary Faculty, Gerbičeva 60, 1000 Ljubljana, Slovenia
Corresponding author. E-mail: jelka.zabavnik@vf.uni-lj.si
Summary: Scrapie is a transmissible degenerative disease of the central nervous system occurring naturally in sheep. It belongs to a group of prion diseases known as transmissible spongiform encephalopathies (TSE) and it is characterized by the accumulation of a proteinase-resistant prion protein mainly in the central nervous system. Three main scrapie-linked polymorphisms in the prion protein gene (Prnp) that modulate susceptibility to scrapie have been described and are located at the codon positions 136, 154 and 171. In order to evaluate and characterize the Prnp polymorphisms in Slovenian autochthonous sheep breeds and to evaluate the genetic susceptibility of these sheep to scrapie, we analyzed the Prnp of 41 sheep of the Istrian Pramenka breed from Slovenia. The polymorphisms at codons 136, 154 and 171 were determined by nucleotide sequencing of the Prnp. Four allelic variants and eight different genotypes were determined. At codon 136, two codon variants were observed, encoding amino acids alanine (A) and valine (V), 82.9 % of the sheep examined had AA, 2.5 % had VV and 14.6 % had AV. At codon 154, 90.2 % had arginine/arginine (RR) and 9.8 % arginine/histidine (RH); at the codon position 171, 46.3 % had amino acids glutamine/glutamine (QQ), 36.6 % had codon variant QR, and 17.1 % had RR codons. The most frequent genotype in the Slovenian population of Istrian Pramenka sheep is ARQ/ARQ (29.3 %). Animals carrying this genotype are moderately susceptible to scrapie, as shown by studies on other sheep breeds that were naturally or experimentally infected. The allelic variant VRQ, known to carry a very high risk of scrapie is only poorly represented in the population of the examined sheep (9.8 %). The more abundant allelic variant was ARR, which is typical to sheep resistant to scrapie (32.9 %).
Key words: Istrian Pramenka; sheep; scrapie; prion protein gene; polymorphism; susceptibility; sequencing
Introduction
Scrapie is a transmissible neurodegenerative disease of sheep and goats, showing characteristic brain pathology with vacuolated neurons, that generally manifests clinically with symptoms like pruritus, motor disorders and body deterioration. It belongs to a group of disorders known as transmissible spongiform encephalopathies (TSE) or prion diseases. Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS) fatal familial insomnia (FFI), fatal sporadic insomnia (FSI) and variant Creutzfeldt-Jakob disease (vCJD) in humans and bovine spongiform encephalopathy (BSE) in cattle also belong to this
Received: July, 2004
Accepted for publication: September, 2004
group. Common to all TSE diseases is the accumulation of an abnormal prion protein (PrPSc) in the tissue of the central nervous system. The normal prion protein (PrPc) is expressed in most tissues of the body, with the highest expression in the nervous tissues. Prions seem to be composed exclusively of a modified isoform of PrP designated PrPSc. The PrPc is converted into PrPSc through a process whereby a portion of its a-helical and coil structure is refolded into p-sheet. This structural transition causes changes in its physico-chemical properties that result in the accumulation of PrPSc in the cells. In its abnormal isoform, the PrPSc is infectious and proteinase-resistant (1, 2).
The occurrence of natural scrapie in sheep is a complex interplay between genetic susceptibility and different strains of the infectious prion (1,
84
J. Zabavnik, M. Cotman, M. Pogacnik, P. Juntes
2, 3). Scrapie-related polymorphisms in the coding region of the prion protein (PrP) gene, Prnp, have been studied in number of sheep breeds in various countries (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). While a range of amino-acid polymorphisms of the sheep Prnp have been described, not all of them have been found to be associated with disease susceptibility. However, the polymorphisms at codon 136 and 171 have been confirmed to be associated with scrapie susceptibility in experimental (10) and natural scrapie; valine at codon 136 and glutamine at codon 171 are often present in the genomes of sheep that are highly susceptible to scrapie (9, 11). For the polymorphism at codon 154, the evidence suggests that histi-dine at codon 154 is associated with a low susceptibility to scrapie in some breeds of sheep (12).
The Istrian Pramenka is a domestic, autochthonous sheep that was traditionally bred in the regions of Istria and the Karst (in Croatia and Slovenia). It originates from primitive domestic sheep known as "Zackel" sheep (13). This milk sheep is very tenacious, its long strong legs, distinct long-stepping walk and its slim snout enables its adaptation and survival in the rough conditions on the rocky terrain in the Karst region. In 2002, the estimated number of breeding females of Istrian Pramenka sheep in Slovenia was 770 animals, but this number is increasing (http://www.tiho-hannover.de/einricht/zucht/ eaap/descript/1617.htm).
The Prnp genotypes of Istrian Pramenka sheep breed have not been examined previously. Therefore, we have studied the Prnp polymorphisms of the Istrian Pramenka that are known to be associated with scrapie susceptibility.
Material and methods
Animals
A total of 41 Istrian Pramenka sheep, from a larger flock of about 400 adult sheep located in Slovenia's Karst region were examined in the study. No clinical signs of scrapie were ever observed in the animals of the examined flock. The absence of the proteinase-resistant prion protein (PrPSc) in the obex tissue of the animals that either died or were slaughtered was confirmed by immunochemical screening using either the rapid post-mortem test Prionics Check Western
(Prionics AG) (western blot analysis) or an ELISA test Enfer TSE Kit (Abbott).
DNA isolation and Prnp amplification
Genomic DNA was isolated from blood leukocytes or from frozen brain tissue by a standard phenol-chloroform protocol as described by Sambrook et al. (18) or with a Wizard Genomic DNA Purification Kit (Promega).
The Prnp, including the whole open reading frame (794 bp), was amplified by a polymerase chain reaction (PCR) with set of primers: SPrP-1 (5' - CATCATGGTGAAAAGC CACATAGGC - 3') and SPrP-2 (5'-GAAAACAGGAAGGTTGCCCCTATCC-3') as described by Ikeda et al. (6). The conditions for the PCR amplification were: initial step at 94 oC for 2 min; denaturation at 94 oC for 1 min; annealing at 58 oC for 1 min; and extension at 72 oC for 2 min. The PCR products were electrophoresed in a 0.8 % agarose gel containing ethidium bromide (all Sigma) and visualised under ultraviolet light.
Sequencing
The PCR products were purified from the agarose gel using Wizard® SV Gel and a PCR Clean-Up System (Promega). The purified PCR products were directly sequenced using an ABI PRISM Dye Terminator Cycle Sequencing Kit with the primers: SPrP5: 5'-ATAAGCTGGGATTCTCTCT-3' SPrP-2: 5'-GAAAACAGGAAGGTTGCCCTATCC-3' as described by Gombojav (14). The sequencing was performed on an ABI Prism 310 autosequencer.
Data analyses
The DNA sequence data were analysed using Chromas (Technelysium).
Results
The western blot analyses and ELISA tests performed on the tissues of the animals that died or were slaughtered were always negative for the proteinase-resistant prion protein (PrP*).
We observed dimorphisms at codons 136, 154 and 171; the different codon frequencies are shown in Table 1. At codon 136 - A and V were observed, at codon 154 - R and H were observed and at codon 171 - Q and R were determined. Four different allelic variants were observed along
Scrapie-susceptibility-linked polymorphisms of the prion protein gene in Istrian Pramenka sheep
85
Table 1: Codon frequencies for Prnp in Istrian Pramenka sheep
Codon position	136			154			171		
Codon variant	AA	AV	W	RR	RH	HH	00	OR	RR
Number	34	6	1	37	4	0	19	15	7
Percentage	82.9	14.6	2.5	90.2	9.8	0	46.3	36.6	17.1
Table 2: Allelic frequencies for Prnp in Istrian Pramenka sheep
Allele (136/154/171)	Number of animals	Percentage
ARQ	43	52.4
ARR	27	32.9
VRQ	8	9.8
AHQ	4	49
Table 3: Genotypes of Prnp in Istrian Pramenka sheep associated with scrapie
susceptibility
Scrapie susceptibility	Number of animals	Percentage
Low (ARR/ARR; ARQ/ARR; AHQ/ARR)	20	48.8
Moderate (ARQ/ARQ; ARQ/AHQ)	14	34.1
High (VRQ/VRQ; ARQ/VRQ)	5	12.2
Occasional occurrence (ARR/VRQ)	2	4.9
with eight different PrP genotypes. Tables 2 and 3 show the allelic and genotypic frequencies of the examined animals. The histidine observed at position 154 was never in the homozygous form.
The frequencies of the genotypes associated with susceptibility to scrapie are shown Figure 1. The most frequent genotype was ARQ/ARQ (29.3 %), which is moderately susceptible to scrapie. Only 12.1 % of Istrian Pramenka population were
shown to be highly susceptible to scrapie (ARQ/VRQ and VRQ/VRQ) (Table 3).
Discussion
The results of our study show the frequencies of different codons for the amino acids at positions 136, 154 and 171 of the PrP protein in the autochthonous Istrian Pramenka sheep breed,
86
J. Zabavnik, M. Cotman, M. Pogacnik, P. Juntes
their allelic variants and genotypes. At codon 136 - A and V were observed, at codon 154 - R and H were observed and at codon 171 - Q and R were determined. As has also been the case for other sheep breeds, not all the theoretically possible allelic and genotypic variants were observed. Despite this, the Istrian Pramenka sheep belongs, in terms of Prnp genetics, to a group of breeds that are genetically complex having four allelic variants and eight different genotypes, similar to Cheviot, Swaledale and Shetland sheep that also have the same four Prnp alleles (19). The most frequent combination of the three codons in the Slovenian population of the Istrian Pramenka was ARQ (52.4 % allelic frequency; Table 2) and the most frequent genotype was ARQ/ARQ (29.3 %; Figure 1). The animals with this genotype are moderately susceptible to scrapie, as was shown by studies on other sheep breeds that were naturally or experimentally infected (9, 10, 11). Fortunately, the allelic variant VRQ, which is known to carry a very high risk of scrapie, was poorly represented in the population of the examined sheep (9.8 %). However, this ratio was still above a recently reported average of 3.7 % from a population of 430233 sheep from 243 different breeds (20). In the examined group of sheep, the homozygous form of the allele was only found in one animal (2.4 %). The allelic variant ARR, which is typical to sheep resistant to scrapie, was better represented in the examined sheep population (32.9 %).
Figure 1: Graphic presentation of Prnp genotype frequencies in the examined Istrian Pra-menka sheep
The genetic data on the Prnp polymorphisms offers the possibility of controlling natural scrapie infections in the sheep population by selecting genotypes carrying scrapie-resistant alleles in breeding animals. Once the genotype is determined it may be possible to breed sheep resistant to scrapie by eliminating the Prnp genes with codon forms 136-V and 171-Q, which have been shown to influence scrapie-susceptibility (21). Breeding programmes for animals carrying genotypes that determine resistance and the elimination of animals with VRQ alleles are already underway in many European countries. The VRQ-allelic variant was poorly represented in the examined sheep breed, therefore its elimination could be possible. However, considering the high frequency of the ARQ-allelic variant in the Istrian Pramenka sheep population, eliminating all the susceptible sheep could be difficult and should be viewed as a long-term process. Additionally, atypical forms of scrapie have also been reported in sheep having genotypes that are known to be resistant to classical scrapie (22). Therefore, consideration should be given to the use of an appropriate breeding programme coupled with the use of diagnostic tests to control the possible onset of scrapie in our population of Istrian Pramenka sheep.
Scrapie in Istrian Pramenka sheep has never been diagnosed in Slovenia. The results of our study show that the majority of the Istrian Pramenka sheep population in Slovenia has the genotype which is moderately susceptible, however
Scrapie-susceptibility-linked polymorphisms of the prion protein gene in Istrian Pramenka sheep
87
some of them also carry the highly susceptible VRQ alleles. Considering the genotypes determined, we could expect the onset of scrapie in the Istrian Pramenka sheep population in Slovenia, therefore special attention should be paid to the monitoring for possible clinical signs of scrapie and to screen any suspect animals for the presence of PrPSc.
Acknowledgements
We thank Mrs. Magdalena Dobravec for her technical assistance. This research was supported by the Slovenian Ministry of Agriculture, Forestry and Food and the Slovenian Ministry of Education, Science and Sport (project number CRP V4-0758-02).
References
1.	Prusiner SB. Prions. Proc Natl Acad Sci USA 1998; 95: 13363-83.
2.	Dalsgaard NJ. Prion diseases. An overview. APMIS. 2002; 110: 3-13.
3.	Tranulis MA. Influence of the prion protein gene, Prnp, on scrapie susceptibility in sheep. APMIS. 2002; 110: 33-43.
4.	Goldmann W, Hunter N, Foster JD, Salbaum JM, Beyreuther K, Hope J. TWo alleles of a neural protein gene linked to scrapie in sheep. Proc Natl Acad Sci USA 1990; 87: 2476-80.
5.	Hunter N, Foster JD, Hope J. Natural scrapie in British sheep: breeds, ages and PrP gene polymorphisms. Vet Rec 1992; 130: 389-92.
6.	Ikeda T, Horiuchi M, Ishiguro N, Muramatsu Y, KaiUwe GD, Shinagawa M. Amino acid polymorphisms of PrP with reference to onset of scrapie in Suffolk and Corriedale sheep in Japan. J Gen Virol 1995; 76: 2577-81.
7.	Belt PB, Muileman IH, Schreuder BE, Bos-de Ruijter J, Gielkens AL, Smits MA. Identification of five allelic variants of the sheep PrP gene and their association with natural scrapie. J Gen Virol 1995; 76: 509-17.
8.	Clouscard C, Beaudry P, Elsen JM, et al. Different allelic effects of the codons 136 and 171 of the prion protein gene in sheep with natural scrapie. J Gen Virol 1995; 76: 2097-101.
9.	Hunter N, Foster JD, Goldmann W, Stear MJ, Hope J, Bostock C. Natural scrapie in a closed flock of Cheviot sheep occurs only in specific PrP genotypes. Arch Virol 1996; 141: 809-24.
10.	O'Rourke KI, Holyoak GR, Clark WW, et al. PrP genotypes and experimental scrapie in orally inoculated Suffolk sheep in the United States. J Gen Virol 1997; 78: 975-8.
11.	Hunter N, Cairns D. Scrapie-free Merino and Poll Dorset sheep from Australia and New Zealand have normal frequencies of scrapie-susceptible PrP genotypes. J Gen Virol 1998; 79: 2079-82.
12.	Thorgeirsdottir S, Sigurdarson S, Thorisson HM, Georgsson G, Palsdottir A. PrP gene polymorphism and natural scrapie in Icelandic sheep. J Gen Virol 1999; 80: 2527-34.
13.	Kavar T, Kompan D, Dovč P. Genetic differentiation among Istrian Pramenka, Bovška Sheep and Jezersko-Solčavska Sheep. Zb Bioteh Fak Univ Ljubl Kmet Zooteh 2002; 2: 193-201.
14.	Gombojav A, Ishiguro N, Horiuchi M, Serjmy-adag D, Byambaa B, Shinagawa M. Amino acid polymorphisms of PrP gene in Mongolian sheep. J Vet Med Sci 2003; 65: 75-81.
15.	Billinis C, Psychas V, Leontides L, et al. Prion protein gene polymorphisms in healthy and scrapie-affected sheep in Greece. J Gen Virol 2004; 85: 54754.
16.	Hunter N, Goldmann W, Benson G, Foster JD, Hope J. Swaledale sheep affected by natural scrapie differ significantly in PrP genotype frequencies from healthy sheep and those selected for reduced incidence of scrapie. J Gen Virol 1993; 74: 1025-31.
17.	EAAP-ANIMAL GENETIC DATA BANK of the , , School of Veterinary Medicine Hannover () (5.7.2004).
18.	Sambrook J, Russell DW. Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2001: 6.4-6.12.
19.	Madelin R. Report on the monitoring and testing of ruminants for the presence of transmissible spongiform encephalopathy (TSE) in the EU in 2003, including the results of the survey of prion protein genotypes in sheep breeds. Brussels: European Commission, 2004.
20.	Report on the monitoring and testing of ruminants for the presence of transmissible spongiform encephalopathy (TSE) in the EU in 2003, including the results of the prion protein genotypes in sheep breeds. European Commission, Health and Consumer Protection Directorate-General. European Communities, May 2004: 80-84.
21.	Perret G, Issaly H, Bouix J, Palhiere I. Taking account of scrapie in the usual genetic improvement scheme of Causses du Lot sheep breed. In: 54th annual meeting of the European Association for Animal Production. Rome, 2003.
22.	Benestad SL, Sarradin P, Thu B, Schonheit J, Tranulis MA, Bratberg B. Cases of scrapie with unusual features in Norway and designation of a new type, Nor98. Vet Rec 2003: 153 (7): 202-8.
88
J. Zabavnik, M. Cotman, M. Pogacnik, P. Juntes
POLIMORFIZMI GENA ZA PRIONSKI PROTEIN, ODGOVORNI ZA DOVZETNOST ZA PRASKAVICO PRI ISTRSKIH PRAMENKAH
J. Zabavnik, M. Cotman, M. Pogačnik, P. Juntes
Povzetek: Praskavica je prenosljiva degenerativna bolezen centralnega živčnega sistema ovac, znana tudi kot oblika prenosljive spongiformne encefalopatije (transmissible spongiform encephalopathy - TSE), s katero se ovce okužijo po naravni poti. Spada v skupino prionskih bolezni, za katere je značilno, da se v celicah kopiči na proteazno delovanje odpornei prion, kar se pojavlja predvsem v centralnem živčnem sistemu. Poznani so trije najpomembnejši polimorfizmi gena za prionski protein (Prnp), ki modulirajo dovzetnost ovac na praskavico; nahajajo se na kodonih 136, 154 in 171. V programu določanja polimor-fizmov gena za PrP in ovrednotenja genetske dovzetnosti slovenskih avtohtonih pasem ovac za praskavico, smo analizirali Prnp pri 41 ovcah pasme istrska pramenka. Polimorfizme na kodonu 136, 154 in 171 smo določali s sekvenciranjem nukleotidnih zaporedij gena Prnp. Ugotovili smo štiri alelne različice in osem genotipov. Na kodonu 136 smo ugotovili dve različici aminokislin, alanin (A) in valin (V). Pri 82,9 % pregledanih živali smo ugotovili alanin/alanin (AA), 2,5 % pregledanih živali je imelo različico VV, 14,6 % pa AV. Na kodonu 154 smo pri 90,2 % ugotovili arginin/arginin (RR), pri 9,8 % živali pa arginin/histidin (RH). Na položaju 171 smo pri 46,3 % preiskovanih živali določili aminokislino glutamin/glutamin (QQ), 36,6 % je imelo različico QR, 17,1 % pa RR. Pri slovenski populaciji istrskih pramenk je najpogosteje zastopan genotip ARQ/ARQ (29,3 %), ki je značilen za ovce, srednje dovzetne za praskavico, kar so pokazale preiskave naravno ali poskusno okuženih ovac. Alelna različica VRQ, ki je značilna za ovce z visokim tveganjem obolevanja za praskavico, je le slabo zastopana v populaciji preiskanih ovac (9,8 %). Bolje je zastopana alelna različica ARR, ki je značilna za živali, odporne na praskavico (32,9 %).
Ključne besede: istrska pramenka; ovca; praskavica; gen za prionski protein; polimorfizem; dovzetnost; sekvenciranje