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Genetic susceptibility to scrapie in a population of Latxa breed sheep in the Basque Country, Spain
Small Ruminant Research 45 (2002) 255–259
Genetic susceptibility to scrapie in a population of Latxa breed sheep in the Basque Country, Spain A. Hurtado∗ , A.L. Garc´ıa-Pérez, I. Beltrán de Heredia, J. Barandika, A. Sanz-Parra, E. Berriatua, R.A. Juste Instituto Vasco de Investigacion y Desarrollo Agrario (NEIKER), Berreaga, 1 48160 Derio, Bizkaia, Spain Received 22 June 2002
Abstract Resistance to natural scrapie in sheep has been associated with polymorphisms at codons 136, 154 and 171 of the prion protein (PrP). This study was undertaken to obtain preliminary information on the PrP gene-associated susceptibility to scrapie in the native sheep breed of the Spanish Basque Country (Latxa), where a single case of scrapie has been reported. Six hundred and ninetyeight sheep from 10 commercial scrapie-free flocks were analysed. The genotype associated with a higher resistance (ARR/ARR) was present in 5.9%, whereas that associated with the highest susceptibility (homozygous VRQ) was absent. ARQ/ARQ was the genotype most frequently observed (48.8%), indicating that susceptible animals exist in the absence of clinical disease. This study provides base-line data for genotype frequencies of Latxa breed that can be included among the criteria used for breed selection to increase the frequencies of resistant genotypes without losing out on other productive characteristics. © 2002 Elsevier Science B.V. All rights reserved. Keywords: PrP genotyping; Scrapie; Sheep
1. Introduction Scrapie is a fatal neurodegenerative disease included in the group of transmissible spongiform encephalopathies (TSEs) that affect sheep and goats. Clinical symptoms include pruritus, excitability, trembling, lack of co-ordination and at a later stage paralysis and death. TSEs are characterised by the accumulation of an abnormal isoform (PrPSc ) of the host prion protein (PrPc ) in the tissues of the CNS and lymphoid tissues of affected individuals. The occurrence of natural scrapie seems to be influenced by polymorphisms at codons 136, 154 and 171 of the ∗ Corresponding author. Tel.: +34-94-4034338, fax: +34-94-4034310. E-mail address: [email protected] (A. Hurtado).
host gene that encodes the PrP (Hunter, 1997). The ARR allele seems to be linked to low risk of scrapie in all breeds and the highest susceptibility has been associated with VRQ, with ARQ as an intermediate situation (Dawson et al., 1998). Selective breeding against scrapie susceptibility may be a useful way of controlling disease incidence and the European Union is encouraging member states to investigate PrP genotype frequencies in European sheep populations (Scientific Steering Committee, 1999). Latxa breed, the native dairy sheep of the Basque Country in Northern Spain, includes ca. 300,000 sheep and 5,000 flocks and two distinct sheep varieties are recognised: the black faced Latxa (BFL) and the fair faced Latxa (FFL) comprising 58 and 42% of the total population, respectively. Whereas, BFL flocks are distributed throughout the Basque Country, FFL
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flocks are restricted to the northeastern area, in the region of Gipuzkoa. Husbandry is traditional, based on keeping the animals on farmland pastures during winter and spring, when lambing takes place (one lambing per ewe per year) and on communal mountain pastures during the summer period. Feeding is based on pasture, except for the winter period (lambing) when concentrate supplementation is added. A milk recording and selection programme (Gabiña et al., 1993) has been in place since the 1980s based on selecting sires to use for artificial insemination (AI) and at present about 35% of the replacements in these flocks are bred by AI. Whereas in Latxa sheep a single case of scrapie has been reported, sheep of almost the same genetic constitution (Manech breed) from the French Atlantic Pyrenees suffer scrapie at a relatively high frequency (Laplanche et al., 1993; Elsen et al., 1997) and disease in Manech red face (MRF) has been associated with ARQ and VRQ alleles (Elsen et al., 1997). The aim of this study was to obtain some preliminary information on the PrP gene-associated susceptibility to scrapie in Latxa breed. There are no similar reports for other breeds in Spain and scrapie in this country is considered a sporadic disease with only 11 cases reported since the first description (Garc´ıa de Jalón et al., 1987), including a single case in a flock of Latxa sheep.
2. Material and methods This PrP genotype study was carried out in 10 flocks (four BFL and six FFL) included in the selection programme, that are currently participating in a European project studying scrapie transmission. Selected flocks have had no cases of scrapie and are located in Gipuzkoa, the province bordering The Atlantic Pyrenees Department of France where the incidence of scrapie in MRF sheep is relatively high (Laplanche et al., 1993). The size of study flocks ranged between 117 and 403 sheep and blood samples with EDTA were collected from about 25% of the animals in each flock (n = 698), including all the rams (n = 68). Genomic DNA was extracted and submitted to a commercial subcontractor (Labogena, France) for PrP polymorphism analysis at codons 136 (alanine (A)/valine (V)), 154 (arginine (R)/histidine
(H)) and 171 (R/Q (glutamine or H)), using restriction fragment length polymorphism analysis of two polymerase chain reaction amplified products (PCR-RFLP) as previously described (Elsen et al., 1999). Briefly, polymorphisms at codons 136 and 154 were determined by BspHI digestion of one of the amplicons and codon 171 polymorphisms (R or Q (glutamine or H)) were detected by BclI restriction analysis of another amplicon (Elsen et al., 1999). Differences in the proportion of alleles and genotypes between flocks, breed varieties or sex were assessed for significance using Chi-squared statistical analysis in EpiInfo 6.4 (CDC, Atlanta). Analysis of the relationship between the presence of scrapie susceptible genotypes and breed variety was carried out using logistic regression in GLIMMIX (SAS institute) to take into account the correlated nature of genotypes in the same flock.
3. Results A total of 698 genotypes (630 ewes and 68 rams) were analysed and the distribution of genotypes in each flock and sheep variety is presented in Table 1. The allele ARQ was the most common (989/1396; 70.8%), followed by ARR (355/1396; 25.4%), VRQ (46/1396; 3.3%) and AHQ (6/1396; 0.4%). Genotypes ARQ/ARQ and ARQ/ARR were identified in 48.8% (341/698) and 37.8% (264/698) of sheep, respectively, ARR/ARR in 5.9% (41/698), ARQ/VRQ in 5.3% (37/698), ARR/VRQ in 1.3% (9/698) and AHQ/ARQ in 0.9% (6/698). The genotype associated to the highest susceptibility (VRQ in homozygosis) was not found in any of the animals analysed. Chi-squared statistical analysis revealed differences in the proportion of alleles and genotypes between flocks or breed varieties (P < 0.05), but not between sexes (P > 0.05). The proportion of scrapie susceptible genotypes ARQ/ARQ and ARQ/VRQ ranged between 74% (37/50) in flock 3 and 37.4% (31/83) in flock 1 (P < 0.01) and was higher among FFL sheep (242/417, 58%) than BFL sheep (136/281, 48%) (P = 0.012). However, further analysis of the relationship between the presence of scrapie susceptible genotypes and breed variety, using logistic regression to take into account the correlated nature of genotypes in the same flock, failed to establish a significant association
A. Hurtado et al. / Small Ruminant Research 45 (2002) 255–259
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Table 1 Frequency distribution of genotypes in each flock and sheep variety BFL
ARR/ARR ARR/ARQ AHQ/ARQ ARQ/ARQ ARR/VRQ ARQ/VRQ No. of examined animals No. of examined rams Flock census
Flock Rams Flock Rams Flock Rams Flock Rams Flock Rams Flock Rams 83 12 361
Total
1
2
3
4
10.8 16.7 50.6 33.3 – – 35.0 41.7 1.2 – 2.4 8.3 49 4 188
– – 42.9 25.0 4.1 – 48.9 75.0 – – 4.1 – 50 4 187
2.0 – 22.0 25.0 2.0 – 74.0 75.0 – – – – 99 6 403
7.0 16.7 51.0 66.6 – – 42.0 16.7 – – – – 281 26
6.0 11.5 44.1 38.5 1.0 – 47.1 46.2 0.4 – 1.4 3.8 90 15 381
FFL 5
6
7
8
9
10
2.2 – 23.4 6.7 – – 55.6 66.6 4.4 6.7 14.4 20 69 4 272
3.0 – 29.0 50.0 1.4 – 58.0 50.0 1.4 – 7.2 – 88 10 369
6.9 10.0 42.0 40.0 – – 50.0 50.0 – – 1.1 – 62 6 245
9.7 – 37.1 33.3 – – 43.5 50.0 1.6 16.7 8.1 – 71 3 295
9.9 – 35.2 66.7 – – 46.5 33.3 2.8 – 5.6 – 37 4 117
2.7 – 37.8 25.0 – – 46.0 50.0 – – 13.5 25.0 417 42
Total
Total Latxa
5.8 2.4 33.6 28.6 0.7 – 50.1 54.7 1.9 4.8 7.9 9.5 698 68
5.9 5.9 37.8 32.3 0.9 – 48.8 51.5 1.3 2.9 5.3 7.4
BFL, black faced Latxa; FFL, fair faced Latxa. In italics frequencies of genotypes for the rams.
between breed variety and occurrence of susceptible genotypes (P > 0.05). 4. Discussion The aim of this study was to provide base-line data on the genetic susceptibility to scrapie in the native breed of the Basque Country by the analysis of 10 Latxa commercial flocks included in the selection programme. According to established criteria for the assessment of susceptibility to scrapie, presence of either Q or H at codon 171 does not seem to affect the level of risk of disease and both are included in the same risk group (Dawson et al., 1998). Therefore, polymorphisms Q or H at codon 171 were not distinguished in this study and were both coded as Q, resulting in a considerable reduction of the cost of genotyping, but without affecting the interpretation of PrP gene-associated susceptibility to scrapie. The ARR allele seems to be linked to low risk of scrapie in all breeds and confers resistance both in homozygosis and heterozygosis (Dawson et al., 1998). In Latxa sheep these represent about 43% of animals. Of these, 14% (5.9% of the total examined sheep) were homozygotes and therefore at a very low risk of scrapie; no cases of scrapie have been described for ARR/ARR sheep in Europe or the USA, although one
case has been reported in a Suffolk sheep in Japan (Ikeda et al., 1995). The other 86% (37% of the total examined sheep) were ARR/ARQ heterozygotes and therefore, at low risk of scrapie in individual sheep, but at varying degrees of risk for progeny depending on genotype of the other parent (Dawson et al., 1998). The VRQ allele has been associated with scrapie, with genotypes VRQ/VRQ and ARQ/VRQ at highest risk (Bossers et al., 1996; Elsen et al., 1999; Tranulis et al., 1999; O’Doherty et al., 2001) and in sheep breeds where VRQ alleles are absent or infrequent, scrapie cases occur in the ARQ/ARQ and ARQ/ARH and rarely in the ARQ/ARR genotypes (Hunter et al., 1997; Elsen et al., 1997). The relatively low frequency of VRQ alleles and high frequency of ARQ alleles and ARQ/ARQ genotypes found in this study indicates that the Latxa population examined could be highly susceptible to scrapie. Overall, the genotype distribution observed in Latxa sheep would closely resemble that described in MRF sheep where incidence of scrapie is associated with ARQ/ARQ and ARQ/VRQ genotypes (Elsen et al., 1997). Differences in the proportion of genotypes between flocks are strongly determined by ram use. Although, AI policy strongly affects this proportion, the present data suggest that the genotype of rams used for natural mating can also influence the genotype frequencies of the flock. For example, the frequency of the VRQ allele among the 15 rams of
258
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flock 5 was significantly higher (23.4%) and consequently the percentage of VRQ for the flock was also higher (9.4%). In the absence of scrapie cases, our results suggest that infective scrapie is not present in these flocks. Previous studies also demonstrated the presence of highly susceptible PrP genotypes among healthy Merino and Poll Dorset sheep (Hunter and Cairns, 1998) and Romney Marsh sheep (Bossers et al., 1999) from scrapie-free countries (Australia and New Zealand). Alternatively, differences in susceptibility to scrapie might be related to other factors such as the scrapie strain present in different areas, which might target different genotypes (Smits et al., 1997), or the level of exposure to infection. These results highlight the need for epidemiological studies aimed at identifying factors that prevent the epidemic spread of scrapie among Latxa sheep from the neighbouring region in France in spite of the close genetics and commercial exchange.
5. Conclusion This is the first report on genetic susceptibility to scrapie in a Spanish breed. Although, clinical disease is extremely rare in Latxa sheep (only a single case ever reported), knowing the PrP genotype of the animals and in particular of the rams used for insemination, is a valuable tool to assess the risk of disease occurrence should the infecting agent be present. Differences between flocks in the presence of scrapie susceptible genotypes are strongly determined by ram use and AI policy. In the future, if data on the genotype distributions of the rams used for AI becomes available, it will be possible to consider changes in the present milk production breeding programme to accommodate selection for scrapie resistance. In this sense the relatively high frequency of scrapie resistant ARQ/ARR and ARR/ARR genotypes in the ewes examined combined with the use of semen from rams with the ARR allele would offer encouraging prospects.
Acknowledgements Financial support was provided by the European Union (Project PL98 7023). A.H. was the recipient of a
Postdoctoral Fellowship from the Instituto Nacional de Investigaciones Agrarias (INIA) of Spain. The authors would like to thank the staff from the Gipuzkoako Foru Aldundiko Abeltzaintza Zerbitzua for collecting blood samples and the farmers for their collaboration.
References Bossers, A., Schreuder, B.E., Muileman, I.H., Belt, P.B., Smits, M.A., 1996. PrP genotype contributes to determining survival times of sheep with natural scrapie. J. Gen. Virol. 77, 2669– 2673. Bossers, A., Harders, F.L., Smits, M.A., 1999. PrP genotype frequencies of the most dominant sheep breed in a country free from scrapie. Arch. Virol. 144, 829–834. Dawson, M., Hoinville, L.J., Hosie, B.D., Hunter, N., 1998. Guidance on the use of PrP genotyping as an aid to the control of clinical scrapie. Scrapie Information Group. Vet. Rec. 142, 623–625. Elsen, J.M., Barillet, F., Vu Tien Khang, J., Schelcher, F., Amigues, Y., Laplanche, J.L., Poivey, J.P., Eychenne, F., 1997. Génetiqué de la sensibilité à la tremblante ovine: recherches en cours et perspectives (genetics of susceptibility to scrapie in sheep: current research and prospects). INRA Prod. Anim. 10, 133– 140. Elsen, J.M., Amigues, Y., Schelcher, F., Ducrocq, V., Andreoletti, O., Eychenne, F., Khang, J.V., Poivey, J.P., Lantier, F., Laplanche, J.L., 1999. Genetic susceptibility and transmission factors in scrapie: detailed analysis of an epidemic in a closed flock of Romanov. Arch. Virol. 144, 431–445. Gabiña, D., Arrese, F., Arranz, J., Beltran de Heredia, I., 1993. Average milk yields and environmental effects on Latxa sheep. J. Diary Sci. 76, 1191–1198. Garc´ıa de Jalón, J.A., De las Heras, M., Balaguer, L., Badiola, J.J., 1987. Enfermedad del prurigo lumbar (scrapie) en la oveja: diagnóstico en 5 rebaños (scrapie disease in sheep: diagnosis in 5 flocks). Med. Vet. 4, 303–312. Hunter, N., 1997. PrP genetics in sheep and the applications for scrapie and BSE. Trends. Microbiol. 5, 331–334. 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, 137–140. 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, 2079– 2082. 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, 2577–2581. Laplanche, J.L., Chatelain, J., Beaudry, P., Dussaucy, M., Bounneau, C., Launay, J.M., 1993. French autochthonous scrapied sheep without the 136Val PrP polymorphism. Mamm. Genome 4, 463–464.
A. Hurtado et al. / Small Ruminant Research 45 (2002) 255–259 O’Doherty, E., Aherne, M., Ennis, S., Weavers, E., Roche, J.F., Sweeney, T., 2001. Prion protein gene polymorphisms in pedigree sheep in Ireland. Res. Vet. Sci. 70, 51–56. Scientific Steering Committee, 1999. Scientific opinion on the policy of breeding and genotyping of sheep, i.e. The issue of whether sheep should be bred to be resistant to scrapie, adopted by the Scientific Steering Committee at its meeting of 22–23 July 1999. European Commission, Directorate-General XXIV,
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Consumer Policy and Customer Health Protection, Scientific Health Opinions (http://europa.eu.int/comm/food/fs/sc/ssc/ out57 en.html). Smits, M.A., Bossers, A., Schreuder, B.E., 1997. Prion protein and scrapie susceptibility. Vet. Q. 19, 101–105. 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, 1073–1077.
Genetic susceptibility to scrapie in a population of Latxa breed sheep in the Basque Country, Spain A. Hurtado∗ , A.L. Garc´ıa-Pérez, I. Beltrán de Heredia, J. Barandika, A. Sanz-Parra, E. Berriatua, R.A. Juste Instituto Vasco de Investigacion y Desarrollo Agrario (NEIKER), Berreaga, 1 48160 Derio, Bizkaia, Spain Received 22 June 2002
Abstract Resistance to natural scrapie in sheep has been associated with polymorphisms at codons 136, 154 and 171 of the prion protein (PrP). This study was undertaken to obtain preliminary information on the PrP gene-associated susceptibility to scrapie in the native sheep breed of the Spanish Basque Country (Latxa), where a single case of scrapie has been reported. Six hundred and ninetyeight sheep from 10 commercial scrapie-free flocks were analysed. The genotype associated with a higher resistance (ARR/ARR) was present in 5.9%, whereas that associated with the highest susceptibility (homozygous VRQ) was absent. ARQ/ARQ was the genotype most frequently observed (48.8%), indicating that susceptible animals exist in the absence of clinical disease. This study provides base-line data for genotype frequencies of Latxa breed that can be included among the criteria used for breed selection to increase the frequencies of resistant genotypes without losing out on other productive characteristics. © 2002 Elsevier Science B.V. All rights reserved. Keywords: PrP genotyping; Scrapie; Sheep
1. Introduction Scrapie is a fatal neurodegenerative disease included in the group of transmissible spongiform encephalopathies (TSEs) that affect sheep and goats. Clinical symptoms include pruritus, excitability, trembling, lack of co-ordination and at a later stage paralysis and death. TSEs are characterised by the accumulation of an abnormal isoform (PrPSc ) of the host prion protein (PrPc ) in the tissues of the CNS and lymphoid tissues of affected individuals. The occurrence of natural scrapie seems to be influenced by polymorphisms at codons 136, 154 and 171 of the ∗ Corresponding author. Tel.: +34-94-4034338, fax: +34-94-4034310. E-mail address: [email protected] (A. Hurtado).
host gene that encodes the PrP (Hunter, 1997). The ARR allele seems to be linked to low risk of scrapie in all breeds and the highest susceptibility has been associated with VRQ, with ARQ as an intermediate situation (Dawson et al., 1998). Selective breeding against scrapie susceptibility may be a useful way of controlling disease incidence and the European Union is encouraging member states to investigate PrP genotype frequencies in European sheep populations (Scientific Steering Committee, 1999). Latxa breed, the native dairy sheep of the Basque Country in Northern Spain, includes ca. 300,000 sheep and 5,000 flocks and two distinct sheep varieties are recognised: the black faced Latxa (BFL) and the fair faced Latxa (FFL) comprising 58 and 42% of the total population, respectively. Whereas, BFL flocks are distributed throughout the Basque Country, FFL
0921-4488/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 4 4 8 8 ( 0 2 ) 0 0 1 5 6 - 6
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flocks are restricted to the northeastern area, in the region of Gipuzkoa. Husbandry is traditional, based on keeping the animals on farmland pastures during winter and spring, when lambing takes place (one lambing per ewe per year) and on communal mountain pastures during the summer period. Feeding is based on pasture, except for the winter period (lambing) when concentrate supplementation is added. A milk recording and selection programme (Gabiña et al., 1993) has been in place since the 1980s based on selecting sires to use for artificial insemination (AI) and at present about 35% of the replacements in these flocks are bred by AI. Whereas in Latxa sheep a single case of scrapie has been reported, sheep of almost the same genetic constitution (Manech breed) from the French Atlantic Pyrenees suffer scrapie at a relatively high frequency (Laplanche et al., 1993; Elsen et al., 1997) and disease in Manech red face (MRF) has been associated with ARQ and VRQ alleles (Elsen et al., 1997). The aim of this study was to obtain some preliminary information on the PrP gene-associated susceptibility to scrapie in Latxa breed. There are no similar reports for other breeds in Spain and scrapie in this country is considered a sporadic disease with only 11 cases reported since the first description (Garc´ıa de Jalón et al., 1987), including a single case in a flock of Latxa sheep.
2. Material and methods This PrP genotype study was carried out in 10 flocks (four BFL and six FFL) included in the selection programme, that are currently participating in a European project studying scrapie transmission. Selected flocks have had no cases of scrapie and are located in Gipuzkoa, the province bordering The Atlantic Pyrenees Department of France where the incidence of scrapie in MRF sheep is relatively high (Laplanche et al., 1993). The size of study flocks ranged between 117 and 403 sheep and blood samples with EDTA were collected from about 25% of the animals in each flock (n = 698), including all the rams (n = 68). Genomic DNA was extracted and submitted to a commercial subcontractor (Labogena, France) for PrP polymorphism analysis at codons 136 (alanine (A)/valine (V)), 154 (arginine (R)/histidine
(H)) and 171 (R/Q (glutamine or H)), using restriction fragment length polymorphism analysis of two polymerase chain reaction amplified products (PCR-RFLP) as previously described (Elsen et al., 1999). Briefly, polymorphisms at codons 136 and 154 were determined by BspHI digestion of one of the amplicons and codon 171 polymorphisms (R or Q (glutamine or H)) were detected by BclI restriction analysis of another amplicon (Elsen et al., 1999). Differences in the proportion of alleles and genotypes between flocks, breed varieties or sex were assessed for significance using Chi-squared statistical analysis in EpiInfo 6.4 (CDC, Atlanta). Analysis of the relationship between the presence of scrapie susceptible genotypes and breed variety was carried out using logistic regression in GLIMMIX (SAS institute) to take into account the correlated nature of genotypes in the same flock.
3. Results A total of 698 genotypes (630 ewes and 68 rams) were analysed and the distribution of genotypes in each flock and sheep variety is presented in Table 1. The allele ARQ was the most common (989/1396; 70.8%), followed by ARR (355/1396; 25.4%), VRQ (46/1396; 3.3%) and AHQ (6/1396; 0.4%). Genotypes ARQ/ARQ and ARQ/ARR were identified in 48.8% (341/698) and 37.8% (264/698) of sheep, respectively, ARR/ARR in 5.9% (41/698), ARQ/VRQ in 5.3% (37/698), ARR/VRQ in 1.3% (9/698) and AHQ/ARQ in 0.9% (6/698). The genotype associated to the highest susceptibility (VRQ in homozygosis) was not found in any of the animals analysed. Chi-squared statistical analysis revealed differences in the proportion of alleles and genotypes between flocks or breed varieties (P < 0.05), but not between sexes (P > 0.05). The proportion of scrapie susceptible genotypes ARQ/ARQ and ARQ/VRQ ranged between 74% (37/50) in flock 3 and 37.4% (31/83) in flock 1 (P < 0.01) and was higher among FFL sheep (242/417, 58%) than BFL sheep (136/281, 48%) (P = 0.012). However, further analysis of the relationship between the presence of scrapie susceptible genotypes and breed variety, using logistic regression to take into account the correlated nature of genotypes in the same flock, failed to establish a significant association
A. Hurtado et al. / Small Ruminant Research 45 (2002) 255–259
257
Table 1 Frequency distribution of genotypes in each flock and sheep variety BFL
ARR/ARR ARR/ARQ AHQ/ARQ ARQ/ARQ ARR/VRQ ARQ/VRQ No. of examined animals No. of examined rams Flock census
Flock Rams Flock Rams Flock Rams Flock Rams Flock Rams Flock Rams 83 12 361
Total
1
2
3
4
10.8 16.7 50.6 33.3 – – 35.0 41.7 1.2 – 2.4 8.3 49 4 188
– – 42.9 25.0 4.1 – 48.9 75.0 – – 4.1 – 50 4 187
2.0 – 22.0 25.0 2.0 – 74.0 75.0 – – – – 99 6 403
7.0 16.7 51.0 66.6 – – 42.0 16.7 – – – – 281 26
6.0 11.5 44.1 38.5 1.0 – 47.1 46.2 0.4 – 1.4 3.8 90 15 381
FFL 5
6
7
8
9
10
2.2 – 23.4 6.7 – – 55.6 66.6 4.4 6.7 14.4 20 69 4 272
3.0 – 29.0 50.0 1.4 – 58.0 50.0 1.4 – 7.2 – 88 10 369
6.9 10.0 42.0 40.0 – – 50.0 50.0 – – 1.1 – 62 6 245
9.7 – 37.1 33.3 – – 43.5 50.0 1.6 16.7 8.1 – 71 3 295
9.9 – 35.2 66.7 – – 46.5 33.3 2.8 – 5.6 – 37 4 117
2.7 – 37.8 25.0 – – 46.0 50.0 – – 13.5 25.0 417 42
Total
Total Latxa
5.8 2.4 33.6 28.6 0.7 – 50.1 54.7 1.9 4.8 7.9 9.5 698 68
5.9 5.9 37.8 32.3 0.9 – 48.8 51.5 1.3 2.9 5.3 7.4
BFL, black faced Latxa; FFL, fair faced Latxa. In italics frequencies of genotypes for the rams.
between breed variety and occurrence of susceptible genotypes (P > 0.05). 4. Discussion The aim of this study was to provide base-line data on the genetic susceptibility to scrapie in the native breed of the Basque Country by the analysis of 10 Latxa commercial flocks included in the selection programme. According to established criteria for the assessment of susceptibility to scrapie, presence of either Q or H at codon 171 does not seem to affect the level of risk of disease and both are included in the same risk group (Dawson et al., 1998). Therefore, polymorphisms Q or H at codon 171 were not distinguished in this study and were both coded as Q, resulting in a considerable reduction of the cost of genotyping, but without affecting the interpretation of PrP gene-associated susceptibility to scrapie. The ARR allele seems to be linked to low risk of scrapie in all breeds and confers resistance both in homozygosis and heterozygosis (Dawson et al., 1998). In Latxa sheep these represent about 43% of animals. Of these, 14% (5.9% of the total examined sheep) were homozygotes and therefore at a very low risk of scrapie; no cases of scrapie have been described for ARR/ARR sheep in Europe or the USA, although one
case has been reported in a Suffolk sheep in Japan (Ikeda et al., 1995). The other 86% (37% of the total examined sheep) were ARR/ARQ heterozygotes and therefore, at low risk of scrapie in individual sheep, but at varying degrees of risk for progeny depending on genotype of the other parent (Dawson et al., 1998). The VRQ allele has been associated with scrapie, with genotypes VRQ/VRQ and ARQ/VRQ at highest risk (Bossers et al., 1996; Elsen et al., 1999; Tranulis et al., 1999; O’Doherty et al., 2001) and in sheep breeds where VRQ alleles are absent or infrequent, scrapie cases occur in the ARQ/ARQ and ARQ/ARH and rarely in the ARQ/ARR genotypes (Hunter et al., 1997; Elsen et al., 1997). The relatively low frequency of VRQ alleles and high frequency of ARQ alleles and ARQ/ARQ genotypes found in this study indicates that the Latxa population examined could be highly susceptible to scrapie. Overall, the genotype distribution observed in Latxa sheep would closely resemble that described in MRF sheep where incidence of scrapie is associated with ARQ/ARQ and ARQ/VRQ genotypes (Elsen et al., 1997). Differences in the proportion of genotypes between flocks are strongly determined by ram use. Although, AI policy strongly affects this proportion, the present data suggest that the genotype of rams used for natural mating can also influence the genotype frequencies of the flock. For example, the frequency of the VRQ allele among the 15 rams of
258
A. Hurtado et al. / Small Ruminant Research 45 (2002) 255–259
flock 5 was significantly higher (23.4%) and consequently the percentage of VRQ for the flock was also higher (9.4%). In the absence of scrapie cases, our results suggest that infective scrapie is not present in these flocks. Previous studies also demonstrated the presence of highly susceptible PrP genotypes among healthy Merino and Poll Dorset sheep (Hunter and Cairns, 1998) and Romney Marsh sheep (Bossers et al., 1999) from scrapie-free countries (Australia and New Zealand). Alternatively, differences in susceptibility to scrapie might be related to other factors such as the scrapie strain present in different areas, which might target different genotypes (Smits et al., 1997), or the level of exposure to infection. These results highlight the need for epidemiological studies aimed at identifying factors that prevent the epidemic spread of scrapie among Latxa sheep from the neighbouring region in France in spite of the close genetics and commercial exchange.
5. Conclusion This is the first report on genetic susceptibility to scrapie in a Spanish breed. Although, clinical disease is extremely rare in Latxa sheep (only a single case ever reported), knowing the PrP genotype of the animals and in particular of the rams used for insemination, is a valuable tool to assess the risk of disease occurrence should the infecting agent be present. Differences between flocks in the presence of scrapie susceptible genotypes are strongly determined by ram use and AI policy. In the future, if data on the genotype distributions of the rams used for AI becomes available, it will be possible to consider changes in the present milk production breeding programme to accommodate selection for scrapie resistance. In this sense the relatively high frequency of scrapie resistant ARQ/ARR and ARR/ARR genotypes in the ewes examined combined with the use of semen from rams with the ARR allele would offer encouraging prospects.
Acknowledgements Financial support was provided by the European Union (Project PL98 7023). A.H. was the recipient of a
Postdoctoral Fellowship from the Instituto Nacional de Investigaciones Agrarias (INIA) of Spain. The authors would like to thank the staff from the Gipuzkoako Foru Aldundiko Abeltzaintza Zerbitzua for collecting blood samples and the farmers for their collaboration.
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