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)
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 pruritus 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 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 tissue 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 primers (PRNP281, 5′-GTCAAGGTGGTAGCCACAGT-3′; and PRNP680, 5′-CCTGGGATTCTCTCTGGTA-3′), as previously described (12). The 25-μL reaction mixture consisted of 1 × 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 μM of each primer (Agriculture & Food Laboratory, Guelph, Ontario), and 2 μL 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 independence and for trend by means of the GraphPad InStat program, 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 submissions 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 Alberta, Ontario, Quebec, and Nova Scotia. The estimated percentages 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.
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 |
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 | 16.7 | 30.6 | 27.4 | 12.3 | 14.2 | 10.3 | 19.5 | 8.9 | |
| AA | RH | 2.5 | 0.6 | 0.4 | 0.4 | 0.0 | 2.3 | 0.8 | 2.4 | ||
| AA | HH | 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 | 2.2 | 3.5 | 1.7 | 2.5 | 1.7 | 0.0 | 1.7 | 0.8 | |
| AV | RH | 0.0 | 0.1 | 0.4 | 0.6 | 0.0 | 1.1 | 0.0 | 0.0 | ||
| VV | RR | 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.
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.
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 haplotypes detected in each province. The VRQ haplotype was much more frequent in Alberta sheep than in sheep from the other provinces. 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 proportion 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%).
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 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 | 15.4 | 12.8 | 18.9 | 14.7 | |
| AA | RH | 0.1 | 0.9 | 0.2 | 0.7 | ||
| AA | HH | 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 | 4.3 | 1.8 | 2.2 | 0.2 | |
| AV | RH | 0.0 | 0.4 | 0.0 | 0.0 | ||
| VV | RR | 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 | |||
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).
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 mutation (wild-type) was also found in a Katahdin male sheep with an ARQ/ARQ genotype.
The haplotype frequencies were similar to the provincial averages 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 averages 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 common 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).
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
| Breed | Total number of sheep | Haplotype; % of annual submissions | Risk group; % of annual submissions | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
|
|
|
||||||||||
| 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 |
One Rideau Arcott sheep with an ARK haplotype is not shown.
Adding the figures for R1 and R2 showed that many breeds were less than 70% resistant (Table V). There were very low numbers for 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 province was ARR, which resulted in the ARR/ARQ and ARR/ARR genotypes being the most frequently observed. The frequency of 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 scrapie, 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 frequency 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 genotyping 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 + R2), a lower proportion of sheep with little resistance (R3), and (with the exception of Alberta) a lower proportion 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 proficiency 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.
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