Prion protein genotypes of sheep as determined from 3343 samples submitted from Ontario and other provinces of Canada from 2005 to 2012

Abstract

This study analyzed sheep prion protein (PrP) genotypes of samples submitted from Ontario and other provinces of Canada to the Animal Health Laboratory at the University of Guelph, Guelph, Ontario, between 2005 and 2012. In Ontario, the proportion of scrapie-resistant sheep increased from 2005 to 2012 as evidenced by an increase in the ARR haplotype. When Canadian provinces (Alberta, Ontario, Quebec, and Nova Scotia) were compared from 2008 to 2012, a high proportion of scrapie-resistant sheep was found in all the provinces. The proportions of resistant sheep were lower in Alberta and Quebec than in Ontario and Nova Scotia. Alberta had higher proportions of susceptible sheep and a higher frequency of VRQ alleles, and Quebec had a higher frequency of the ARQ allele.

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Article
260 The Canadian Journal of Veterinary Research 2014;78:260–266
Introduction
Scrapie is a transmissible spongiform encephalopathy that affects
sheep and goats. Based on the clinical presentation, scrapie can be
classified into classic and atypical forms. Classic scrapie typically
presents with behavior changes, ataxia, incoordination, and pruri-
tus that may affect multiple younger sheep (2 to 4 y of age) within
a flock, whereas atypical scrapie tends to occur as single cases
without pruritus within older sheep (1,2). There is no treatment for
scrapie, and the disease is always fatal. Several codons of the sheep
prion protein (PrP), including codons 136, 154, and 171, have been
identified as important determinants of an individual’s resistance
to classic scrapie. The 136A/154R/171R haplotype (ARR) confers
resistance, homozygous individuals being the most resistant (3–5).
Alleles ARQ, AHQ, ARH, and VRQ are associated with increased
risk for the development of scrapie (1,6,7).
In North America the Canadian Food Inspection Agency (CFIA)
and the United States Department of Agriculture (USDA) consider
scrapie genotyping based on analysis of PrP codons 136 and 171
as a tool that can be used in an overall plan to manage the risk of
scrapie on a particular premises. For comparison with published
data, this study considered the system used by the National Scrapie
Plan for Great Britain (8), which has classified PrP genotypes on the
basis of PrP codons 136, 154, and 171 into 5 risk groups (R1 to R5)
according to susceptibility to scrapie. The most resistant individuals
are in group R1 (ARR/ARR); those in group R2 (ARR-containing
genotypes) also have a high degree of resistance. Group R3 sheep
have little resistance, and sheep in groups R4 and R5 are considered
susceptible to scrapie. Selective breeding programs have been an
effective means of scrapie control. The Ram Genotyping Scheme
in Britain (negative selection for VRQ, positive selection for ARR
genotypes) has resulted in significant increases in the ARR haplotype
and R1 sheep, with reductions in other haplotypes and susceptible
sheep (R3 to R5) (8). In scrapie-affected flocks, increasing the ARR
haplotype through breeding results in decreased scrapie risk at the
population level; the opposite holds true for other alleles such as
AHQ, VRQ, ARH, and ARQ (7,9,10).
To date, the number of studies describing PrP genotypes of
domestic sheep in Canada is limited. In 3 Canadian flocks with a
history of scrapie a relatively low proportion of highly resistant R1
sheep (11.1%) was found in a 1998–2008 study, most sheep falling
into risk groups R2 (42.1%) and R3 (40.4%) and the remainder into
groups R4 (2.1%) and R5 (4.3%) (6). A study of Quebec rams from
49 flocks in 2003 found an overall higher level of resistance, with
the following distribution: R1, 21.8%; R2, 49.0%; R3, 23.8%; R4, 2.8%;
and R5, 2.6% (11). In comparison, 319 341 sheep genotyped as part
Prion protein genotypes of sheep as determined from 3343 samples
submitted from Ontario and other provinces of Canada from 2005 to 2012
Colin Cameron, Patricia Bell-Rogers, Rebeccah McDowall, Ana R. Rebelo, Hugh Y. Cai
Abstract
This study analyzed sheep prion protein (PrP) genotypes of samples submitted from Ontario and other provinces of Canada to
the Animal Health Laboratory at the University of Guelph, Guelph, Ontario, between 2005 and 2012. In Ontario, the proportion
of scrapie-resistant sheep increased from 2005 to 2012 as evidenced by an increase in the ARR haplotype. When Canadian
provinces (Alberta, Ontario, Quebec, and Nova Scotia) were compared from 2008 to 2012, a high proportion of scrapie-resistant
sheep was found in all the provinces. The proportions of resistant sheep were lower in Alberta and Quebec than in Ontario
and Nova Scotia. Alberta had higher proportions of susceptible sheep and a higher frequency of VRQ alleles, and Quebec had
a higher frequency of the ARQ allele.
Résumé
Dans la présente étude les génotypes de la protéine prion du mouton (PrP) d’échantillons en provenance de l’Ontario et d’autres provinces
canadiennes soumis au Animal Health Laboratory de l’Université de Guelph, Ontario, entre 2005 et 2012 ont été analysés. En Ontario, la
proportion de moutons résistants à la tremblante a augmentée entre 2005 et 2012 tel que démontré par une augmentation de l’haplotype
ARR. Lorsque les provinces canadiennes (Alberta, Ontario, Québec, et Nouvelle-Écosse) ont été comparées de 2008 à 2012, des proportions
élevées de moutons résistants à la tremblante ont été trouvés dans toutes les provinces. Les proportions de moutons résistants étaient plus
faibles en Alberta et au Québec qu’en Ontario ou en Nouvelle-Écosse. L’Alberta avait une proportion plus élevée de moutons susceptibles
et une fréquence plus élevée d’allèles VRQ, et le Québec une fréquence plus élevée de l’allèle ARQ.
(Traduit par Docteur Serge Messier)
Animal Health Laboratory, University of Guelph, Guelph, Ontario N1G 2W1.
Address all correspondence to Dr. Hugh Y. Cai; telephone: 519-836-4120, ext. 54316; fax: 519-821-8072; e-mail: hcai@uoguelph.ca
Received April 18, 2013. Accepted September 30, 2013.

2000;64:0–00 The Canadian Journal of Veterinary Research 261
of the Compulsory Scrapie Flock Scheme in Great Britain from 2005
to 2007 had much higher levels of resistance and fewer sheep with
little resistance, the proportions being as follows: R1, 31.2%; R2,
41.8%; R3, 17.8%; R4, 4.1%; and R5, 5.1% (10). From this it appears
as though Canadian sheep may have lower resistance to scrapie or
simply that representative published data in this area are lacking.
This study analyzed sheep PrP genotypes of samples submitted
from Ontario and other provinces of Canada to the Animal Health
Laboratory at the University of Guelph, Guelph, Ontario, between
2005 and 2012.
Materials and methods
Samples
Sheep whole blood (in ethylenediamine tetraacetic acid) or tis-
sue samples (ear notches) were submitted to the Animal Health
Laboratory at the University of Guelph for scrapie PrP genotyping
between 2005 and 2012. The samples were submitted by veterinarians
on behalf of producers for diagnostic purposes without solicitation.
DNA extraction
Sample genomic DNA was extracted from whole blood with a
Roche MagNA Pure LC instrument and the MagNA Pure LC DNA
Isolation Kit I (Roche Diagnostics, Laval, Quebec), an Applied
Biosystems MagMax-96 and the MagMAX Pathogen RNA/DNA Kit
(Life Technologies, Burlington, Ontario), or a DNeasy Blood & Tissue
Kit (Qiagen, Mississauga, Ontario). Ear-notch DNA was extracted
with the MagNA Pure LC DNA Isolation Kit I or the DNeasy Blood &
Tissue Kit. For MagNA Pure extractions the tissue was homogenized
with a stainless steel bead in TriPure (Roche) on a bead mixer mill
(Retsch, Newtown, Pennsylvania, USA), and then extraction was
done with the MagNA Pure LC instrument.
Real-time polymerase chain reaction (RT-PCR)
melt-curve genotyping
Genotyping RT- PCR was done with the use of commercially
available kits (TIB MolBiol, Adelphia, New Jersey, USA) according
to the manufacturer’s instructions. These kits use 3 combinations of
probes that measure
fluorescent resonance energy transfer (
FRET
)
specific to the regions of the PrP gene that correspond to codons
136, 154, and 171. Mutations in the PCR products are detected by
measuring the melting temperature of PCR product–probe hybrids.
The PCR reactions, done with either the Roche LightCycler 2.0 or the
Roche LightCycler 480II instrument, were followed by melt-curve
analysis to determine genotype at codons 136, 154, and 171. When
the melt-curve peaks did not match known or common genotypes,
direct sequencing of the PCR products was done.
Direct sequencing
For samples requiring direct sequencing, a PCR product of 399 base
pairs was obtained from the PRNP gene with the use of specific prim-
ers (PRNP281, 5’-GTCAAGGTGGTAGCCACAGT-3’; and PRNP680,
5’-CCTGGGATTCTCTCTGGTA-3’), as previously described (12). The
25-mL reaction mixture consisted of 1 3 GeneAmp PCR Gold Buffer,
2.5 mM of MgCl2, 0.1 mM of deoxynucleotide triphosphates, 0.75 U
of AmpliTaq Gold DNA polymerase (Life Technologies), 0.2 mM of
each primer (Agriculture & Food Laboratory, Guelph, Ontario), and
2 mL of template DNA. Amplification was done on a BioMetra T3
thermocycler (Montreal Biotech, Dorval, Quebec) with denaturation
at 95°C for 12 min followed by 35 cycles of amplification (at 94°C for
20 s, 55°C for 30 s, and 72°C for 60 s) and a final extension at 72°C
for 7 min. After confirmation of a PCR product of the correct size
by agarose gel electrophoresis the PCR products were sequenced
at the Agriculture & Food Laboratory with the use of both forward
and reverse primers. Sequence electropherograms were assembled
and analyzed by means of SeqMan software (DNASTAR, Madison,
Wisconsin, USA) to determine the PrP genotype of the sample.
Statistical analysis
Percentage comparison was done with Chi-square tests for inde-
pendence and for trend by means of the GraphPad InStat pro-
gram, version 3.06 for Windows (GraphPad Software, San Diego,
California, USA).
Results
Included in the analysis of Ontario sheep from 2005 to 2012 were
73 unique flocks, a minimum of 12 and a maximum of 28 within any
year. A total of 2222 Ontario sheep were genotyped from 2005 to
2012, annual totals ranging from 87 to 488. As low numbers of sub-
missions were received from other Canadian provinces until 2008,
comparisons between provinces were done using only the data from
2008 to 2012, which were obtained for 3343 samples submitted from
Table I. Frequencies of prion protein (PrP) haplotypes at codons 136, 154, and 171 in samples from Ontario sheep submitted
to the Animal Health Laboratory, University of Guelph, Guelph, Ontario, from 2005 to 2012
% of annual submissions
Haplotype 2005 2006 2007 2008 2009 2010 2011 2012
ARR 47.1 41.3 47.5 56.5 56.3 63.8 55.5 69.9
ARQ 39.1 52.9 49.0 35.5 37.9 30.5 40.7 28.0
AHQ 3.8 2.1 1.5 2.0 0.0 1.7 1.3 1.2
ARH 7.8 0.5 0.2 1.8 3.3 1.7 0.0 0.0
VRQ 2.2 3.2 1.9 4.2 2.5 1.7 2.1 0.8
ARK 0.0 0.0 0.0 0.0 0.0 0.6 0.4 0.0

262 The Canadian Journal of Veterinary Research 2000;64:0–00
Alberta, Ontario, Quebec, and Nova Scotia. The estimated percent-
ages of the total sheep population (as of January 1, 2012; Statistics
Canada; www.statcan.gc.ca/pub/23-011-x/23-011-x2011002-eng.
pdf) included in this study from each province in 2008 to 2012 were
0.46%, 0.38%, 0.4%, and 1.12%, respectively. Few submissions were
received from other provinces, and these were not included in
the analysis. The samples received from 2008 to 2012 represented
23 sheep breeds, the most common being Suffolk, Rideau Arcott,
Polled Dorset, Romanov, Canadian Arcott, Hampshire, and Texel.
Ontario (2005–2012)
The PrP haplotype with the highest frequency in Ontario sheep
was ARR; next most frequent was ARQ (Table I). Over the study
period the frequency of ARR increased, from about 40% to 50% in
2005–2007 to about 55% to 70% in 2008–2012. Small decreases in
the ARQ, AHQ, and ARH haplotypes were observed to account
for this increase. The VRQ haplotype appeared to be relatively
stable. The ARK allele was detected only twice, in individuals
with 136AA/154RR/171QK and 136AV/154RR/171QK genotypes.
Overall, there was a trend to an increase in the proportion of resistant
genotypes (risk groups 1 and 2) between 2005 and 2012 (P , 0.0001)
(Table II, Figure 1). This appears to be due to an increase in the
number of ARR- homozygous individuals (R1) and a decrease in
the number of R3 individuals during this period. No major changes
were observed in the proportion of R2, R4, or R5 sheep.
Figure 1. Frequency distribution by risk for scrapie of Ontario sheep
genotyped at the Animal Health Laboratory, University of Guelph, Guelph,
Ontario, between 2005 and 2012 according to the classification system
of the National Scrapie Plan for Great Britain (8). The most resistant
individuals are in group R1 (ARR/ARR); those in group R2 (ARR-containing
genotypes) also have a high degree of resistance. Group R3 sheep
have little resistance, and sheep in groups R4 and R5 are considered
susceptible to scrapie.
Table III. Frequencies by province of PrP haplotypes at codons
136, 154, and 171 in samples from Canadian sheep submitted
to the Animal Health Laboratory from 2008 to 2012
% of annual submissions
Haplotype AB ON QC NS
ARR 55.5 58.8 54.7 59.9
ARQ 37.0 35.0 42.3 35.2
AHQ 0.1 1.5 0.3 2.1
ARH 0.0 1.5 0.1 1.1
VRQ 7.5 3.0 2.6 1.7
ARK 0.0 0.1 0.0 0.0
AB — Alberta; ON — Ontario; QC — Quebec; NS —Nova Scotia.
Table II. Genotypes for PrP at codons 136, 154, and 171 of Ontario sheep as a percentage of annual number of samples submitted
to the Animal Health Laboratory
Codon % of annual submissions
Scrapie risk group 136 154 171 2005 2006 2007 2008 2009 2010 2011 2012
R1 AA RR RR 24.4 18.8 26.6 29.3 29.2 42.5 33.9 52.0
R2 AA RR QR 34.9 40.3 40.7 43.0 45.0 36.8 39.8 35.0
AA RH QR 2.2 1.2 0.4 2.7 0.0 0.0 1.7 0.0
AA RR RH 6.2 1.0 0.0 3.3 5.8 3.4 0.0 0.0
R3 AA RR QQ 16.7 30.6 27.4 12.3 14.2 10.3 19.5 8.9
AA RH QQ 2.5 0.6 0.4 0.4 0.0 2.3 0.8 2.4
AA HH QQ 0.7 1.2 0.8 0.2 0.0 0.0 0.0 0.0
AA RR HH 1.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0
AA RR QH 5.1 0.0 0.4 0.4 0.8 0.0 0.0 0.0
AA RH QH 1.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0
R4 AV RR QR 2.2 2.5 0.8 5.3 3.3 2.3 1.7 0.8
R5 AV RR QQ 2.2 3.5 1.7 2.5 1.7 0.0 1.7 0.8
AV RH QQ 0.0 0.1 0.4 0.6 0.0 1.1 0.0 0.0
VV RR QQ 0.0 0.1 0.4 0.0 0.0 0.0 0.0 0.0
NC AA RR QK 0.0 0.0 0.0 0.0 0.0 1.1 0.0 0.0
AV RR QK 0.0 0.0 0.0 0.0 0.0 0.0 0.8 0.0
Total number of sheep 275 770 241 488 120 87 118 123
Number of flocks 12 28 18 22 13 12 15 14
R1 — the most resistant; R2 — high degree of resistance; R3 — little resistance; R4 and R5 — susceptible; NC — haplotype not classified for
scrapie risk.

2000;64:0–00 The Canadian Journal of Veterinary Research 263
Canadian provinces (2008–2012)
As with Ontario, the most frequent PrP haplotype in the other
Canadian provinces included in the analysis for the years 2008 to
2012 was ARR, and next most frequent was ARQ (Table III). These
2 haplotypes combined accounted for 92% or more of all the hap-
lotypes detected in each province. The VRQ haplotype was much
more frequent in Alberta sheep than in sheep from the other prov-
inces. Nova Scotia had the highest proportion of ARR-homozygous
(R1) individuals and Alberta and Quebec the lowest proportions
(Table IV). About 80% of all sheep tested in Ontario (79.7%) and
Nova Scotia (80.4%) had resistant (R1 or R2) genotypes (Figure 2);
the resistant proportions were somewhat lower in Alberta (70.4%)
and Quebec (75.9%). There was a trend to an increase in the propor-
tion of resistant genotypes (risk groups 1 and 2) in Ontario between
2005 and 2012 (P , 0.0001). Homozygous ARQ individuals (R3) were
more frequent in Quebec (18.9%) than in the other provinces (12.8%
to 15.4%). A higher proportion of sheep belonged to the R4 and R5
risk groups in Alberta (14.0%) as compared with Ontario (5.9%),
Quebec (5.0%), and Nova Scotia (3.3%).
Mutations for PrP at codons other than 136, 154, and 171 were also
observed. A change in 2012 to a new PrP genotyping kit with the
ability to detect mutations at codon 141 (wild-type is LL) resulted in
the identification of 13 sheep with the 141FL mutation and 1 sheep
with the 141FF mutation (9 from Ontario and 5 from Quebec, from
a variety of breeds) out of 1205 samples (1.16%). One 137TT muta-
tion (wild-type) was also found in a Katahdin male sheep with an
ARQ/ARQ genotype.
The haplotype frequencies were similar to the provincial aver-
ages for most sheep breeds, the most common haplotypes being
ARR and ARQ (Table V). There were, however, some differences
observed with certain breeds. Horned Dorset and Polled Dorset
had relatively higher ARR frequency and lower ARQ frequency:
76%.0 ARR and 24% ARQ for Horned Dorset and 66.6% ARR and
28.5% ARQ for Polled Dorset. Breeds displaying somewhat lower
ARR and higher ARQ frequencies compared with provincial aver-
ages included Hampshire (50.0% ARR and 48.7% ARQ), Southdown
(41.4% ARR and 58.6% ARQ), and crossbreeds (42.2% ARR and
52.9% ARQ). Other breeds, such as Texel (lower ARQ) and North
Country Cheviot (lower ARR), had higher frequencies of less com-
mon haplotypes (AHQ, ARH, and VRQ) as compared with other
breeds. High VRQ frequency was observed in several breeds. A
few breeds with higher numbers (. 50) had somewhat high VRQ
frequencies (. 3%): Canadian Arcott (15.0%), Polled Dorset (4.5%),
North Country Cheviot (9.2%), Romanov (4.1%), and crossbreeds
(3.9%). Black Welsh Mountain sheep had frequencies higher than
average for haplotypes associated with both resistance (68.8% ARR)
and susceptibility (31.3% VRQ).
Adding the figures for R1 and R2 showed that many breeds were
less than 70% resistant (Table V). There were very low numbers for
Figure 2. Frequency distribution by risk for scrapie of Canadian sheep
grouped by province (AB — Alberta; ON — Ontario; QC — Quebec; NS —
Nova Scotia) genotyped at the Animal Health Laboratory between 2008
and 2012. There was a trend to an increase in the proportion of resistant
genotypes (R1 and R2) in Ontario between 2005 and 2012 (P , 0.0001).
Table IV. Genotypes for PrP at codons 136, 154, and 171 of Canadian sheep grouped by province as
a percentage of annual number of samples submitted to the Animal Health Laboratory from 2008
to 2012
Codon % of annual submissions
Scrapie risk group 136 154 171 AB ON QC NS
R1 AA RR RR 31.7 34.1 31.0 36.4
R2 AA RR QR 38.7 41.2 44.3 39.1
AA RH QR 0.0 1.6 0.5 3.6
AA RR RH 0.0 2.8 0.1 1.3
R3 AA RR QQ 15.4 12.8 18.9 14.7
AA RH QQ 0.1 0.9 0.2 0.7
AA HH QQ 0.0 0.1 0.0 0.0
AA RR QH 0.0 0.3 0.1 0.9
R4 AV RR QR 8.8 3.7 2.6 3.1
R5 AV RR QQ 4.3 1.8 2.2 0.2
AV RH QQ 0.0 0.4 0.0 0.0
VV RR QQ 0.9 0.0 0.2 0.0
NC AA RR QK 0.0 0.1 0.0 0.0
AV RR QK 0.0 0.1 0.0 0.0
Total number of sheep 669 936 1290 448

264 The Canadian Journal of Veterinary Research 2000;64:0–00
many of these breeds except for North Country Cheviot (n = 60) and
Canadian Arcott (n = 183). For the major breeds (those with . 100
individuals tested), including Polled Dorset, Hampshire, Rideau
Arcott, Romanov, Suffolk, and Texel, more than 70% of individuals
were resistant. The only major breed with less than 70% resistance
was Canadian Arcott, at 62.0%.
Discussion
The current study suggests an overall high level of resistance to
classic scrapie in Canadian sheep flocks according to PrP genotypes
at codons 136, 154, and 171. Selection for resistant PrP genotypes in
Ontario sheep appears to have resulted in increased resistance in
this province since 2005. Some differences existed between Canadian
provinces during the period 2008 to 2012. Sheep in Ontario and Nova
Scotia appear to have had slightly higher resistance to scrapie than
those in Alberta and Quebec.
The increase in resistance to scrapie in Ontario sheep from 2005
to 2012 is a result of the increased frequency of the ARR haplotype
(and R1 genotype) during this period. This increase is related to a
decrease in R3 genotypes over the same period, mainly as a result
of decreases in AHQ and ARH haplotypes. Interestingly, certain PrP
haplotypes and genotypes associated with increased scrapie risk
remained relatively stable throughout the study. The VRQ haplotype,
generally considered to be the most susceptible to scrapie, was stable
throughout the study. The VRQ haplotype accounted for 1.7% to
4.2% of all haplotypes, and the total frequency of VRQ-containing
genotypes (R4 and R5) ranged from 3.3% to 8.4%. This genotype
frequency was somewhat variable but was without any clear trend
from 2005 to 2012.
Selection for resistant individuals and, in particular, resistant rams
has been associated with a reduction in the incidence of scrapie in
sheep flocks (7). This type of selection has also been demonstrated
to be without significant effect on milk production, reproductive
traits, growth, or carcass traits (8,13–15). These are the reasons for
adoption of programs such as the National Scrapie Plan for Great
Britain, in which negative selection for VRQ and positive selection
for ARR alleles among rams is encouraged. Under this program from
2002 to 2006 the frequency of the ARR allele increased while that of
all other haplotypes decreased, and these changes were reflected
in the changes in scrapie risk groupings: increased R1, relatively
unchanged R2, and decreased R3, R4, and R5 (8). These changes in
British sheep are similar to the pattern observed in Ontario sheep
during this study.
When Canadian provinces were compared for the years 2008 to
2012, the haplotype with the highest frequency within each prov-
ince was ARR, which resulted in the ARR/ARQ and ARR/ARR
genotypes being the most frequently observed. The frequency of
Table V. Distribution of haplotypes and scrapie risk groups by sheep breed among samples submitted from Alberta, Ontario,
Quebec, and Nova Scotia to the Animal Health Laboratory for PrP genotyping analysis between 2008 and 2012
Total number Haplotype; % of annual submissions Risk group; % of annual submissions
Breed of sheep ARR ARQ AHQ ARH VRQ R1 R2 R3 R4 R5
Border Leicester 10 30.0 5.0 45.0 0.0 20.0 10.0 30.0 20.0 10.0 30.0
Black Welsh Mountain 24 68.8 0.0 0.0 0.0 31.3 41.7 0.0 0.0 54.2 4.2
Canadian Arcott 183 51.4 33.6 0.0 0.0 15.0 24.6 38.3 9.3 15.3 12.6
Clun Forest 7 92.9 7.1 0.0 0.0 0.0 85.7 14.3 0.0 0.0 0.0
Charollais 10 60.0 40.0 0.0 0.0 0.0 50.0 20.0 30.0 0.0 0.0
Horned Dorset 25 76.0 24.0 0.0 0.0 0.0 56.0 40.0 4.0 0.0 0.0
Dorper 5 0.0 40.0 0.0 0.0 60.0 0.0 0.0 20.0 0.0 80.0
Polled Dorset 235 66.6 28.5 0.2 0.2 4.5 45.5 34.9 10.6 7.2 1.7
East Friesian 10 76.0 24.0 0.0 0.0 0.0 0.0 80.0 20.0 0.0 0.0
Finnish Landrace 2 25.0 75.0 0.0 0.0 0.0 0.0 50.0 50.0 0.0 0.0
Hampshire 159 50.0 48.7 0.0 0.0 1.3 23.3 53.5 20.8 0.0 2.5
Icelandic 3 0.0 83.3 0.0 0.0 16.7 0.0 0.0 66.7 0.0 33.3
Katahdin 2 25.0 75.0 0.0 0.0 0.0 0.0 50.0 50.0 0.0 0.0
North Country Cheviot 60 42.5 41.7 3.3 3.3 9.2 21.7 28.3 31.7 13.3 5.0
Newfoundland Local 3 16.7 50.0 0.0 0.0 33.3 0.0 0.0 33.3 33.3 33.3
Polypay 91 59.3 38.5 1.6 0.0 0.5 37.4 42.9 18.7 1.1 0.0
Rideau Arcotta 362 53.0 46.1 0.4 0.1 0.1 30.7 44.8 24.3 0.0 0.0
Rambouillet 1 0.0 100.0 0.0 0.0 0.0 0.0 0.0 100.0 0.0 0.0
Romanov 184 59.2 36.7 0.0 0.0 4.1 35.9 42.9 13.0 3.8 4.3
Shetland 4 25.0 50.0 0.0 0.0 25.0 0.0 25.0 25.0 25.0 25.0
Southdown 70 41.4 58.6 0.0 0.0 0.0 10.0 62.9 27.1 0.0 0.0
Suffolk 771 61.5 36.8 0.0 0.0 1.8 38.3 43.7 14.5 2.7 0.8
Texel 136 59.6 25.7 5.1 9.6 0.0 27.9 63.2 8.8 0.0 0.0
Cross 51 42.2 52.9 1.0 0.0 3.9 19.6 41.2 31.4 3.9 3.9
a One Rideau Arcott sheep with an ARK haplotype is not shown.

2000;64:0–00 The Canadian Journal of Veterinary Research 265
the R1 genotype (31.0% to 36.4%) is considerably higher than that
reported in other studies of Canadian sheep (6,11). This difference
may be due to differences in study design or timing. The study by
Harrington and colleagues (6) was limited to a small number of
scrapie-infected flocks, and the study by L’Homme, Leboeuf, and
Cameron (11) was done in 2003. Sheep in the Canadian provinces
included in the study had a high level of resistance to classic scra-
pie, the sum of groups R1 and R2 being greater than 70%. A similar
proportion of resistant genotypes was observed among sheep in
Great Britain (8,10,16).
Lower proportions of scrapie-resistant sheep were observed in
Alberta and Quebec. In Quebec sheep, this difference was a result
of a higher frequency of the ARQ haplotype, ARQ/ARQ genotype,
and R3 individuals than in the other provinces. Ontario, Quebec, and
Nova Scotia had similar percentages of R4 and R5 sheep. In contrast,
Alberta had higher proportions of R4 and R5 sheep compared with
the other provinces. This difference is a result of a much higher fre-
quency of the VRQ haplotype in Alberta, which suggests a greater
susceptibility to classic scrapie in this province.
Published data on province- or country-wide PrP genotyp-
ing analysis of Canadian sheep are not available. The Scrapie
Canada National Genotyping Survey began in 2005 with the goal
of genotyping Canadian sheep and increasing resistance to scrapie.
According to Scrapie Canada (17), 9300 sheep were genotyped
between 2005 and 2009, and the proportions of sheep belonging to
the various risk groups were as follows: R1, 24%; R2, 40%; R3, 26%;
R4, 5%; and R5, 5%. In comparison, the current study of Canadian
provinces between 2008 and 2012 showed that all provinces had
a higher proportion of R1 sheep, a higher proportion of scrapie-
resistant sheep (R1 1 R2), a lower proportion of sheep with little
resistance (R3), and (with the exception of Alberta) a lower propor-
tion of susceptible sheep (R4 and R5). These differences between
the 2 studies may indicate a trend to increased genetic resistance to
scrapie over time in these provinces.
Differences in the frequency of PrP haplotypes and in resistance
to scrapie have been observed between certain breeds of sheep
(16,18–20). In the current study, some differences were observed.
When the most common breeds (those with . 100 individual
samples: Canadian Arcott, Polled Dorset, Hampshire, Rideau Arcott,
Romanov, Suffolk, and Texel) were considered, all except Canadian
Arcott were found to be highly resistant. A high ARQ and VRQ
frequency in Canadian Arcott sheep resulted in a high proportion
(48.6%) of susceptible individuals. A relatively high VRQ haplotype
frequency (7.1%) was also observed in another study of Canadian
sheep (11). Texel sheep had higher frequencies of the AHQ and ARH
haplotypes, resulting in a higher percentage of R2 individuals within
this breed. High frequencies of these haplotypes have previously
been observed in the Texel breed (16,18). Many of the less common
breeds had haplotype frequencies or risk-group proportions that
were considerably different from the averages for all the sheep.
However, making inferences about scrapie resistance within these
breeds on the basis of these low numbers is not practical. In the
analysis of all sheep across provinces, a lower level of resistance to
scrapie was observed in Alberta and Quebec compared with Ontario
and Nova Scotia. There is a possibility that this difference is the
result of differences in breed distribution among the provinces. Some
breeds having less than 70% resistance were found exclusively or
in higher proportions in Alberta (4.9% Black Welsh Mountain and
18.3% Canadian Arcott) and in Quebec (6.7% Canadian Arcott and
4.5% crossbred sheep) than in other provinces (data not shown). In
addition, other highly resistant breeds were found in Ontario but not
in Alberta or Quebec. Breed information was not included with many
samples submitted during this study. In particular, it was provided
with only 22.1% of the samples from Nova Scotia, compared with
more than 70% of the samples from the other provinces.
Certain rare PrP mutations were observed at codons 171 and 137.
The rare ARK haplotype was observed only twice in Ontario (in 2010
and 2011). These individuals had ARQ/ARK (no breed information)
and VRQ/ARK (Rideau Arcott) genotypes and were from the same
flock. The effect of this haplotype on susceptibility to scrapie has not
been fully studied, but a report of a scrapie-positive sheep with the
genotype ARK/ARH suggests that it may not confer resistance (21).
A single mutation at codon 137 was also observed, in an ARQ/ARQ
Katahdin ram that was homozygous for threonine [137TT; versus
wild-type homozygous methionine (137MM)]. This mutation has
been described in various breeds (18,22,23). In a study of classic
scrapie outbreaks in Sarda sheep the AT137RQ haplotype was found
to be protective (23).
Another well-described mutation at codon 141 of the PrP protein
from leucine (L) to phenylalanine (F) was observed on a number of
occasions, with an overall frequency of 1.16%. The presence of this
mutation did not appear to be related to any specific breed of sheep,
as it was identified in both Dorset and North Country Cheviot. The
genotypes of the identified sheep included 8 ARQ/ARR, 4 ARQ/
ARQ, 1 ARQ/AHQ, and 1 ARQ/VRQ. The 141F mutation did appear
to be related to the ARQ haplotype, as described previously (19,24),
and has been associated with an increased risk for atypical/Nor98
scrapie, a form of scrapie that is different from classic scrapie and
was first identified in Norway in 1998 (25–27). The transmission of
atypical scrapie has been demonstrated (28,29) but is still not fully
understood. Cases of atypical scrapie have since been confirmed in
the United States and Canada (30,31). According to the CFIA, 11 of
49 confirmed cases of scrapie in Canada since 2005 were identified
as atypical scrapie. This number of cases could be an underestimate,
as differences in tissue PrPSc accumulation and diagnostic methods
may result in false-negative results (32). Given the incidence of
atypical scrapie in Canada (31), further investigation into the link
between the 141F mutation and genetic resistance to atypical scrapie
is warranted.
In summary, there appears to be a high level of resistance to classic
scrapie in sheep from all the Canadian provinces included in this
study, Ontario and Nova Scotia having a slightly higher proportion
of resistant genotypes than Alberta and Quebec.
Acknowledgments
The authors thank Shruti Patel and Hamid Haghighi for technical
assistance, Dr. Aru Balachandran for providing advice and profi-
ciency samples for method development, and Dr. Beverly McEwen
for assistance with statistical analysis. Partial funding for this study
was provided by the Ontario Ministry of Agriculture, Food and Rural
Affairs through the Animal Health Strategic Investment.

266 The Canadian Journal of Veterinary Research 2000;64:0–00
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