1 National Reference Centre for Transmissible Spongiform Encephalopathies, Animal Pathology Department, Veterinary Faculty, C/Miguel Servet 177, 50013 Zaragoza, Spain
2 Biochemical Genetics and Blood Groups Laboratory, University of Zaragoza, C/Miguel Servet 177, 50013 Zaragoza, Spain
3 Institute for Animal Health, Neuropathogenesis Unit, West Mains Road, Edinburgh EH9 3JF, UK
Correspondence
Cristina Acín
crisacin{at}unizar.es
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
These authors contributed equally to this work.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
At present, little is known about PrP haplotype distribution in Spanish sheep; Hurtado et al. (2002) have described the haplotype frequencies observed for this gene in Latxa sheep. The Rasa Aragonesa breed is a white, polled, thin-tailed, medium-wool sheep (Mason, 1991
). Its direct ancestor was Ovis aries ligeriensis and it remained as a native breed (without foreign interbreeding) until the early 19th century. During the last two centuries, the Rasa Aragonesa has been genetically mixed with other sheep, mainly Merino, and now forms a relatively heterogeneous group (Altarriba & Lamuela, 1983
). It now represents the second most common Spanish breed, after the Merino sheep, at 16·2 % of all Spanish sheep.
The first scrapie case detected in Spain was in 1987 (García de Jalón et al., 1987) and by 2001 there has been an increase in the number of scrapie-affected sheep flocks. In this study, we have analysed four natural scrapie-affected sheep flocks, belonging to Rasa Aragonesa and Rasa Navarra breeds that were diagnosed in the Spanish National Reference Centre for TSE between 2001 and 2003.
The aim of this study was to determine the PrP polymorphisms in Spanish sheep, with special regard to scrapie incidence in the Rasa-related population. Genotypic frequencies observed in the scrapie-affected animals were compared with those detected in healthy sheep from these flocks. In addition, 16 healthy flocks belonging to Rasa Aragonesa, Ojinegra, Cartera, Maellana, Roya Bilbilitana, Ansotana and Churra Tensina breeds were also analysed in order to determine the genetic scrapie risk of these native populations. All these breeds are considered as derivative breeds from the ancient O. aries ligeriensis and are bred in the same geographical region, contributing to possible breed admixtures.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Scrapie-affected sheep.
The total number of scrapie-affected sheep samples was 38. Twenty-three were from the flocks of the casecontrol study and 15 were from additional archival material (paraffin-embedded tissue) of scrapie cases diagnosed in the Spanish National Reference Centre for TSE.
Native healthy flocks.
Blood samples from native breeds (n=905) were obtained from 16 healthy flocks that belong to Rasa Aragonesa (n=296; 4 flocks), Ojinegra (n=182; 4 flocks), Cartera (n=136; 2 flocks), Maellana (n=115; 2 flocks), Roya Bilbilitana (n=96; 2 flocks), Churra Tensina (n=32; 2 flocks) and Ansotana (n=48; 1 flock) breeds. All reproductive rams were sampled from each flock, except from the Ansotana flock, where only the ewes were available. As this breed is endangered, we obtained the samples from a conservation flock.
DNA extraction
Blood.
Genomic DNA was extracted using the GFX genomic blood DNA purification kit (Amersham Pharmacia Biotech).
Paraffin-embedded brain.
Sections of 10 µm were placed in a sterile 1·5 ml tube and 1 ml xylene was added and vortexed vigorously to remove the paraffin. The samples were incubated in a shaker for 15 min at room temperature and the tissue was pelleted by centrifugation at 13 250 g for 5 min, followed by the removal of the supernatant. These steps were repeated once. The pellet was washed in 1 ml 100 % ethanol and spun at 13 250 g for 5 min. The supernatant was removed and the tissue was dried for 15 min at 60 °C. Tissues were immersed in a solution of 180 µl ATL buffer (Qiagen) and 20 µl proteinase K (200 µg ml1; Qiagen). After vortexing vigorously for 1 min, the sections were incubated overnight at 56 °C. After overnight digestion, DNA was purified using a standard phenol/chloroform/isoamyl alcohol (25 : 24 : 1) treatment. The ethanol-precipitated DNA pellet was dissolved in 50 µl TE buffer (Qiagen).
PrP genotype analysis.
Amino acids are described in the single letter code. The codon position is given after the letter; polymorphisms are shown for example as A136V. Haplotypes are presented as groups of amino acids in the order of their codons, whereby positions 136, 154 and 171 have no codon identification, whereas other dimorphisms are identified by a codon position following the letter (in superscript) i.e. A136-R154-R171 (ARR), A136-F141-R154-Q171 (AF141RQ). Genotypes are presented as combinations of the haplotype codes as described above, i.e. ARR/AF141RQ.
RFLP analysis.
All blood and paraffin samples except for the RAS2 and RAS3 flocks were analysed using this methodology. Polymorphisms at codons 136 and 154 were detected by PCR-RFLP analysis using the restriction enzyme BspHI (New England Biolabs) as described by O'Doherty et al. (2000). Polymorphism at codon 171 was detected by two allele-specific PCR amplifications and further digestion with BslI for R171 and AccI for H171 allele detection as described by Yuzbasiyan-Gurkan et al. (1999)
. The presence of Q171 was deduced indirectly from the absence of the expected restriction fragments. During the course of this study, it became apparent that novel polymorphisms at codon 171 (i.e. K171) or close to codon 171 (i.e. D172) interfered with this RFLP analysis. Therefore, additional sequence analysis for genotype determination was used. When DNA from paraffin-embedded tissues was used as a template, an increase to 35 amplification cycles was necessary for PCR-RFLP analysis.
Sequencing.
The complete PrP open reading frame has been sequenced in 697 sheep including 34 scrapie-affected sheep, the complete scrapie-affected flocks RNS, RAS2 and RAS3, 46 % of RAS1, 50 % of Ojinegra sheep, 30 % of Rasa Aragonesa, 25 % of Cartera, 20 % of Maellana, 25 % of Roya Bilbilitana, 30 % of Ansotana and 20 % of Churra Tensina. The whole coding region was amplified using the following primers: 20fwd, 5'-ATGGTGAAAAGCCACATAGGCAGT-3' (codons 18) and 767rev, 5'-CTATCCTACTATGAGAAAAATGAG-3' (250stop codon). PCR fragments were purified using the MALDIspot kit and the vacuum manifold from Millipore and sequenced with the Big Dye kit from Applied Biosystems. The same PCR primers were used for bi-directional sequencing and chromatograms were analysed using BioEdit v.4.8.6 (Hall, 1999).
Statistical analysis.
The genotype distributions obtained for the different populations were compared statistically using the 2 test for independence and the Yates correction for continuity for NxK contingency tables. P<0·05 was considered statistically significant. Haplotypic and genotypic frequencies for each population were calculated using the GENEPOP program (Raymond & Rousset, 1995
). This program was also used to carry out a statistical test to determine possible deviations from the HardyWeinberg proportion. A Markov-chain method was applied to calculate exact P-values; the length of the chain was set to 100 000 iterations. Some flocks showed HardyWeinberg disequilibrium, probably because of non-random mating common in domestic populations. The
2 statistic treating alleles (or haplotypes) rather than genotypes as individual entities, is only appropriate when the HardyWeinberg equilibrium holds (Sasieni, 1997
). As some of the analysed populations were not in equilibrium, we decided to analyse genotypic data. In order to avoid a possible flock effect, before pooling together the data for breed comparison, the distribution of genotypic frequencies was compared between each flock belonging to the same breed using the
2 test for independence and the Yates correction for continuity for NxK contingency tables.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Four-flock casecontrol study.
Scrapie cases: 23 sheep with clinical scrapie signs were sacrificed and the diagnosis was confirmed for all of them. Four animals were from the RNS flock and all showed the homozygous ARQ/ARQ genotype. The other 19 sheep were from the Rasa Aragonesa flocks (15 from RAS1, two from RAS2 and two from RAS3), with 18 sheep of ARQ/ARQ genotype and only one of ARR/ARQ genotype. The frequency of 95·5 % for ARQ/ARQ genotype sheep in this study was not significantly different from the 93 % of the same genotype in the retrospective study and therefore all scrapie-affected animals (n=38) have been combined into one group for haplotype frequency comparison.
Healthy animals: six PrP haplotypes, ARQ, ARR, AHQ, ARH, VRQ and ARK, were found in these four flocks. The ARQ haplotype is the most common in all four flocks, with frequencies between 66·2 and 78·7 % (Table 1). The ARR haplotype is the second most common (14·525·7 %), and the AHQ, ARH and VRQ haplotypes were detected at low frequencies (AHQ 05·4 %, ARH 0·66·1 % and VRQ 0·64·7 %). The rare ARK variant was observed in one heterozygous sheep from the RAS3 flock.
|
By comparison, the haplotype frequencies for the other four PrP variants were considerably lower (VRQ 0·313·5 %, AHQ 07·6 %, ARH 09·4 % and ARK 1 %). Table 1
shows the frequencies obtained for each breed. By means of the PCR-RFLP technique, it is not possible to discriminate between the ARQ and ARK variants (see Methods), which may lead to incorrect estimates of the ARQ frequency if the ARK haplotype is present. Sequencing of 100 ARQ/ARQ sheep from the 182 Ojinegra samples confirmed only four heterozygous ARK/ARQ animals. The ARK haplotype was not found in 197 sheep sequences from the other six breeds in this survey (26 % of total). Assuming that the selection of sequencing samples from the total was random, we estimate that the ARK frequency is 04 %. The impact of the genotyping method on the ARQ frequency is therefore very small.
Statistical differences were not observed in the genotype distribution between the flocks that belong to the same breed, except for the Maellana population. The two Maellana flocks have been considered independently, whereas data from the remaining flocks that belong to the same breed have been pooled together. The differences observed between populations allow the classification of these breeds into different groups (Table 1). First, a group that contains the Rasa Aragonesa, Ojinegra, Roya Bilbilitana and Maellana (only flock 2) breeds, where the most common genotype is ARQ/ARQ and the susceptible VRQ variant is only present in low frequencies (<3 %). The second group includes the Ansotana, Churra Tensina and Maellana (only flock 1) breeds, with intermediate frequencies for the ARQ/ARQ genotype and relatively high frequencies for VRQ. A third group, which in this study only contains the Cartera population, was different from any other breed because of the very high frequency of the resistance-associated ARR/ARQ and ARR/ARR genotypes (66 %).
Association of PrP genotypes with natural scrapie.
The frequency of the ARQ/ARQ genotype in scrapie-affected animals was considerably higher than in the healthy sheep from RAS2 and RNS scrapie-affected flocks (P<0·05) and the frequency of ARQ/ARQ was not significantly different between the scrapie-free and scrapie-affected animals in the RAS1 and RAS3 flocks. Genotype frequencies in healthy animals from scrapie-affected flocks and the native breed group containing Rasa Aragonesa, Ojinegra and Roya Bilbilitana were similar, whereas the frequency for the genotype ARQ/ARQ was significantly higher in the scrapie-affected animals than the frequency values obtained for the second and third groups. We can therefore conclude that the ARQ/ARQ genotype confers risk of scrapie infection to the Spanish sheep breeds. The scrapie-affected sheep showed low variability, with only four observed genotypes (ARQ/ARQ, ARR/ARQ, AR143RQ/ARQ and R101ARQ/ARQ; see Table 3).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nearly 95 % of the scrapie cases in Rasa breeds were homozygous ARQ/ARQ and the frequency of this genotype was significantly higher in scrapie animals than in healthy sheep. This is unexpected, as our analysis revealed about 20 animals with the VRQ haplotype in healthy flockmates. The VRQ haplotype is generally classified as a high-risk factor for scrapie infection (Hunter et al., 1993, 1994
, 1996
; Thorgeirsdottir et al., 1999
). However, our results are similar to studies of so-called alanine breeds' (sheep breeds with no or only occasional VRQ carriers), where scrapie cases occur in the ARQ/ARQ, ARQ/ARH genotypes and rarely in the ARR/ARQ genotype (Hunter et al., 1997
; Elsen et al., 1999
). Therefore, it remains to be established whether the PrP haplotype risk classification for Spanish sheep breeds needs to be different from the classification used for UK sheep breeds.
The survey of the native breeds showed a similar genotype distribution for the Rasa Aragonesa (in both, scrapie and healthy flocks), Ojinegra and Roya Bilbilitana flocks. The haplotype frequencies in these breeds resembled the frequencies published for Suffolk and Lacaune sheep, in which the VRQ haplotype is absent or its frequency is very low (Westaway et al., 1994; Clouscard et al., 1995
; Ikeda et al., 1995
; Hunter et al., 1997
). The detected haplotype frequencies are also similar to those reported for the Spanish Latxa sheep (Hurtado et al., 2002
).
Two native populations (Churra Tensina and Ansotana) could be considered as belonging to the so-called valine breeds' (sheep breeds with a significant number of VRQ carriers; Hunter et al., 1996). The haplotype and genotype frequencies are similar to the values reported for other European, Australian and New Zealand valine breeds' (Hunter et al., 1997
; Hunter & Cairns, 1998
; Thorgeirsdottir et al., 1999
; Drögemüller et al., 2001
). In these breeds, VRQ is associated with a very high risk of scrapie disease and healthy homozygote VRQ/VRQ sheep of old age in scrapie-affected sheep flocks are rare. Sheep of these breeds are less likely to be affected if they are heterozygous at codon 154 (Q/H) or codon 171 (Q/R) (Hunter et al., 1996
). In this survey, the AHQ haplotype was only found at low frequencies (Table 1
). However, the ARR haplotype was found at relative high frequency in these two flocks; they also showed the highest frequency in all flocks of the ARR/VRQ genotype. Besides presenting susceptible genotypes, no scrapie case has been reported in the Churra Tensina and Ansotana breeds, which may reflect the possibility that they have not come into contact with the scrapie agent. Genetically susceptible sheep are present in scrapie-free areas, such as New Zealand and Australia (Hunter et al., 1997
), and can stay healthy after import into countries with high incidence of scrapie, such as the UK, if they are not brought into contact with the scrapie agent (Hunter & Cairns, 1998
). It will therefore be of major importance to investigate the epidemiology of Spanish scrapie cases in conjunction with the genetic profiling presented here. The Churra Tensina and Ansotana breeds are close to extinction, and the application of a scrapie eradication programme based on the susceptible VRQ and ARQ haplotypes could reduce the effective breeding population to a dangerously low level, with serious consequences for the survival of the breed. However, it should be possible as a first step to eliminate VRQ-carrying rams from breeding and to increase the number of ARR carriers.
The sequencing analysis has revealed the complexity of PrP genetics of these native sheep populations. In addition to the known polymorphisms, we have detected five more amino acid changes, but most of these new polymorphisms have very low frequencies (<5 %) and all of them seem to have originated from a mutation in an ancestral (ARQ) haplotype. It is tempting to speculate that the codon 151 polymorphism, resulting in an amino acid substitution of arginine with glycine or histidine, could have a similar association with disease as has been described for the adjacent codon 154 dimorphism (Goldmann et al., 1991). An equivalent polymorphism (R148H) has recently been found in chimpanzees (Soldevila et al., 2004
). The ARQD172 and ARQE175 haplotypes are of special interest as the amino acid changes are very close to the disease-associated codon position 171. Whether they have a similar disease effect remains to be established. Only the R101ARQ and AR143QR haplotypes were present in scrapie-affected animals, but their frequency was not significantly different from expectation. Four of the new dimorphisms have not been found in other species and the codon 151175 region of PrP is N-terminal to the region linked to human prion diseases such as familial CreutzfeldtJacob disease and GerstmannSträusslerScheinker syndrome (GSS) (Windl et al., 1999
). However, codon 101 (equivalent to codon 98 in human PrP) is adjacent to two genetic mutations (P102L and P105L) in human GSS and is therefore of special interest.
In conclusion, we report here the high variability of the PrP gene found in Spanish sheep. The genotype distribution indicates that the breeds that form large populations in Spain, such as the Ojinegra and Rasa Aragonesa, could be highly susceptible to scrapie. Although a larger population study needs to be conducted based on these PrP genotype frequencies, the application of a breeding programme to control scrapie appears to be a challenging task. The aim of most breeding programmes is to control scrapie susceptibility by the gradual elimination of haplotypes associated with high scrapie susceptibility and to encourage the use of breeding rams with the ARR/ARR genotype. The Rasa and Ojinegra populations present ARR/ARR genotype frequencies lower than 5 % and heterozygous frequencies are around 25 %, presenting ARQ in rather high frequencies. Therefore, it may be important to establish the properties of some of the new PrP variants, as they may provide selectable resistance-associated haplotypes. This paper has shown the importance of a full, sequence-based genotype study before making recommendations for the most suitable sheep-breeding programme.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Belt, P. B., Muileman, I. H., Schreuder, B. E., Bos-de Ruijter, J., Gielkens, A. L. & Smits, M. A. (1995). Identification of five allelic variants of the sheep PrP gene and their association with natural scrapie. J Gen Virol 76, 509517.[Abstract]
Clouscard, C., Beaudry, P., Elsen, J. M. & 7 other authors (1995). Different allelic effects of the codons 136 and 171 of the prion protein gene in sheep with natural scrapie. J Gen Virol 76, 20972101.[Abstract]
Drögemüller, C., Leeb, T. & Distl, O. (2001). PrP genotype frequencies in German breeding sheep and the potential to breed for resistance to scrapie. Vet Rec 149, 349352.[Medline]
Elsen, J. M., Amigues, Y., Schelcher, F. & 7 other authors (1999). Genetic susceptibility and transmission factors in scrapie: detailed analysis of an epidemic in a closed flock of Romanov. Arch Virol 144, 431445.[CrossRef][Medline]
García de Jalón, J. A., De las Heras, M., Balaguer, L. & Badiola, J. J. (1987). Enfermedad del prúrigo lumbar (scrapie) en la oveja: diagnóstico en 5 rebaños. Medicina Veterinaria 4, 56 (in Spanish).
Goldmann, W., Hunter, N., Benson, G., Foster, J. D. & Hope, J. (1991). Different scrapie-associated fibril proteins (PrP) are encoded by lines of sheep selected for different alleles of the Sip gene. J Gen Virol 72, 24112417.[Abstract]
Gombojav, A., Ishiguro, N., Horiuchi, M., Serjmyadag, D., Byambaa, B. & Shinagawa, M. (2003). Amino acid polymorphisms of PrP gene in Mongolian sheep. J Vet Med Sci 65, 7581.[CrossRef][Medline]
Guo, X., Kupfer, D. M., Fitch, G. Q., Roe, B. A. & DeSilva, V. (2003). Identification of a novel lysine-171 allele in the ovine prion protein (PRNP) gene. Anim Genet 34, 303305.[CrossRef][Medline]
Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/NT. Nucleic Acids Symp Ser 41, 9598.
Hunter, N. (2000). Transmissible spongiform encephalopathies. In Breeding for Disease Resistance in Farm Animals, pp. 325339. Edited by R. F. E. Axford, S. C. Bishop, F. W. Nicholas & J. B. Owen. Wallingford, UK: CABI.
Hunter, N. & Cairns, D. (1998). Scrapie-free Merino and Poll Dorset sheep from Australia and New Zealand have normal frequencies of scrapie susceptible PrP genotypes. J Gen Virol 79, 20792082.[Abstract]
Hunter, N., Goldmann, W., Benson, G., Foster, J. D. & Hope, J. (1993). 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 74, 10251031.[Abstract]
Hunter, N., Goldmann, W., Smith, G. & Hope, J. (1994). The association of a codon 136 PrP gene variant with the occurrence of natural scrapie. Arch Virol 137, 171177.[Medline]
Hunter, N., Foster, J. D., Goldmann, W., Stear, M. J., Hope, J. & Bostock, C. (1996). Natural scrapie in a closed flock of Cheviot sheep occurs only in specific PrP genotypes. Arch Virol 141, 809824.[Medline]
Hunter, N., Goldmann, W., Foster, J. D., Cairns, D. & Smith, G. (1997). Natural scrapie and PrP genotype: case-control studies in British sheep. Vet Rec 141, 137140.[Medline]
Hurtado, A., Garcia-Perez, A. L., Beltran de Heredia, I., Barandika,J.,Sanz-Parra, A., Berriatura, E. & Juste, R. A. (2002). Genetic susceptibility to scrapie in a population of Latxa breed sheep in the Basque Country, Spain. Small Rumin Res 45, 255259.[CrossRef]
Ikeda, T., Horiuchi, M., Ishiguro, N., Muramatsu, Y., Kai-Uwe, G. D. & Shinagawa, M. (1995). Amino acid polymorphisms of PrP with reference to onset of scrapie in Suffolk and Corriedale sheep in Japan. J Gen Virol 76, 25772581.[Abstract]
Laplanche, J. L., Chatelain, J., Westaway, D., Thomas, S., Dussaucy,M., Brugere-Picoux, J. & Launay, J. M. (1993). PrP polymorphisms associated with natural scrapie discovered by denaturing gradient gel electrophoresis. Genomics 15, 3037.[CrossRef][Medline]
Mason, I. L. (1991). Genetics Resources of Pig, Sheep and Goat. Amsterdam: Elsevier Science.
O'Doherty, E., Aherne, M., Ennis, S., Weavers, E., Hunter, N., Roche,J. F. & Sweeney, T. (2000). Detection of polymorphisms in the prion protein gene in a population of Irish Suffolk sheep. Vet Rec 146, 335338.[Medline]
Raymond, M. & Rousset, F. (1995). GENEPOP (version 1.2): population genetics software for exact test and ecumenicism. J Hered 86, 248249.
Sasieni, P. D. (1997). From genotypes to genes: doubling the sample size. Biometrics 53, 12531261.[Medline]
Smits, M. A., Bossers, A. & Schreuder, B. E. C. (1997). Prion protein and scrapie susceptibility. Vet Q 19, 101105.[Medline]
Soldevila, M., Andres, A. M., Blancher, A., Calafell, F., Ordoñez, M., Pumarola, M., Oliva, B., Aramburu, J. & Bertranpetit, J. (2004). Variation of the prion gene in chimpanzees and its implication for prion diseases. Neurosci Lett 355, 157160.[CrossRef][Medline]
Stephens, A., Wansg, S., Holyoak, G. R., Timofeevskaia, O., Shay,T.L., Vernon, W., Ellis, S., Beever, J. & Cockett, N. (1998). Characterization of the Prion Protein (PrP) Gene in Ten Breeds of Sheep. Proceedings of the Plant & Animal Genome VI Conference, 1822 January 1998.
Thorgeirsdottir, S., Sigurdarson, S., Thorisson, H. M., Georgsson,G. & Palsdottir, A. (1999). PrP gene polymorphism and natural scrapie in Icelandic sheep. J Gen Virol 80, 25272534.
Tranulis, M. A., Osland, A., Bratberg, B. & Ulvund, M. J. (1999). Prion protein gene polymorphisms in sheep with natural scrapie and healthy controls in Norway. J Gen Virol 80, 10731077.[Abstract]
Vaccari, G., Petraroli, R., Agrimi, U. & 8 other authors (2001). PrP genotype in Sarda breed sheep and its relevance to scrapie. Arch Virol 146, 20292037.[CrossRef][Medline]
Westaway, D., Zuliani, V., Cooper, C. M., Da Costa, M., Neuman, S., Jenny, A. L., Detwiler, L. & Prusiner, S. B. (1994). Homozygosity for prion protein alleles encoding glutamine-171 renders sheep susceptible to natural scrapie. Genes Dev 8, 959969.[Abstract]
Windl, O., Giese, A., Schulz-Schaffer, W. & 7 other authors (1999). Molecular genetics of human prion diseases in Germany. Hum Genet 105, 244252.[CrossRef][Medline]
Yuzbasiyan-Gurkan, V., Krehbiel, J. D., Cao, Y. & Venta, P. J. (1999). Development and usefulness of new polymerase chain reaction-based test for detection of different alleles at codons 136 and 171 of the ovine prion protein gene. Am J Vet Res 60, 884887.[Medline]
Received 19 February 2004;
accepted 22 April 2004.