Acta Veterinaria Hungarica 56 (2), pp. 173–180 (2008) DOI: 10.1556/AVet.56.2008.2.4

BREEDING FOR SCRAPIE RESISTANCE IN THE HUNGARIAN SHEEP POPULATION László FÉSÜS1*, Attila ZSOLNAI1, István ANTON1 and László SÁFÁR2 1

Research Institute for Animal Breeding and Nutrition, H-2053 Herceghalom, Gesztenyés út 1, Hungary; 2Hungarian Sheepbreeders’ Association, Budapest, Hungary (Received 16 May 2007; accepted 6 September 2007)

The first results of the Hungarian sheep prion protein (PrP) genotyping programme are discussed in this paper. To obtain initial genotype frequency data 10 commercial (Hungarian Merino, German Mutton Merino, Merino Landschaf, German Blackheaded, Suffolk, Texel, Ile de France, Charollais, Lacaune, British Milksheep) and 4 indigenous (Gyimes Racka, Hortobágy Racka, Tsigaja, Cikta) breeds were sampled in 2003 and 2004, and the PrP genotypes were determined by microsequencing analysis with capillary electrophoresis. In all commercial breeds, a higher number of sheep were genotyped in 2005 (3648) and in 2006 (3834) within the breeding programme to increase scrapie resistance, and the estimated frequency data were compared to the initial figures to evaluate the efficiency of selection. The new developments arising from the identification of the so-called ‘atypical’ scrapie cases are also discussed. Key words: Scrapie, sheep, prion protein genotypes, marker-assisted selection

Transmissible spongiform encephalopathies (TSEs) are a group of progressive neurological disorders. The agent causing TSEs is most probably an abnormal form of a cell-surface glycoprotein called prion (PrP). During the disease this protein accumulates in the body, first of all in the central nervous system. TSEs affect a number of animal species and humans, and include scrapie of sheep and goats and bovine spongiform encephalopathy (BSE). A variant form of Creutzfeldt-Jakob disease (vCJD) of humans was described in 1996 and is *

Corresponding author; E-mail: [email protected]; Phone: 0036 (23) 319-133 0236-6290/$ 20.00 © 2008 Akadémiai Kiadó, Budapest

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thought to have been the result of BSE-infected cattle tissues entering the human food chain. Since sheep may also have been exposed to BSE-contaminated meat and bone meal (Butler, 1998) and BSE can be experimentally transmitted to sheep (Foster et al., 1993; Jeffrey et al., 2001; Houston et al., 2003), the possible human risk of consuming BSE-infected sheep meat has prompted research into this area. In sheep, polymorphisms at codons 136, 154 and 171 of PrP are closely linked to susceptibility to scrapie (Hunter et al., 1992, 1994; Belt et al., 1995). Based on this linkage, a classification system of five risk groups (NSP1 to NSP5) has been established under the National Scrapie Plan (NSP) in the UK (DEFRA, 2004), with NSP1 indicating a very low, and NSP5 a very high risk of scrapie infection, and a genotyping programme to increase scrapie resistance has been started. The possible relationships between scrapie and BSE have led the European Union to the decision of establishing breeding programmes aimed at selection for resistance to scrapie in sheep breeds in all member states (European Commission, 2003). Hungary has a sheep population of 1.2 million. Several commercial and four native, indigenous breeds are represented; more than 90 per cent of the population are Merino or Merino-type sheep. There are approximately 7500 sheep farmers in the country, the average herd size being 150 breeding ewes. The main source of income is light-weight slaughter lamb production for export. The last clinical scrapie case in Hungary was diagnosed in 1964 (Áldásy and Süveges, 1964); the small infected flock was stamped out. In December 2004 the general assembly of the Hungarian Sheepbreeders’ Association accepted the modifications of the existing breeding programme in accordance with Decision 2003/100 of the European Commission. Since an active sheep and goat scrapie surveillance programme is being conducted in the country, seven scrapie cases have been reported in sheep. The aim of the present study is to describe the breeding programme, to present prion protein (PrP) genotype frequency data for the commercial and indigenous breeds, and to evaluate the preliminary results of the breeding programme. Materials and methods Ten commercial (Hungarian Merino, German Mutton Merino, Merino Landschaf, German Blackheaded, Suffolk, Texel, Ile de France, Charollais, Lacaune, British Milksheep) and four indigenous (Gyimes Racka, Hortobágy Racka, Tsigaja, Cikta) breeds were included in the study. In all breeds, to obtain initial frequency data, a small number of sheep, sampled in several flocks, were genotyped in 2003 and 2004. A higher number of commercial sheep were genotyped in the breeding programme in 2005 (n = 3648) and 2006 (n = 3834).

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Genomic DNA to be analysed was isolated from whole blood: 50 μl of blood was washed three times with 500 μl wash solution (10 mM Tris-HCl, pH 7.5, 1 mM Na2EDTA, pH 8.0) and centrifuged at 12,000 rpm in an Eppendorf tube, then 100 μl of lysis solution (10 mM Tris, pH 7.5, 50 mM KCl, 0.5% Tween 20, 0.6 μg/μl Proteinase-K) was added to the pellet. It was shaken for 1 h at 56 °C, then the protease was inactivated by incubating the solution at 94 °C for 10 min. This lysis solution was used as a substrate in the first PCR reaction. The primers used for the amplification of the part of prion gene (266 bp) covering the mutations (1st PCR) were CATTTGCTCCACCACTCGCT and TTGGTGGCTACATGCTGGGAA. The first PCR reaction contained 0.2 μM of primers, 200 μM dNTPs, 1.5 mM MgCl2, 0.5 U DyNAzyme polymerase (Finnzymes, Oy, Espoo, Finland) and 1 μl of sample prepared as described above, in 40 μl volume (10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100). The temperature profile of the reaction was the following: 94 °C for 1 min, 30 cycles with 94 °C for 30 s, 58 °C for 30 s, 72 °C for 30 s. At the end of the 30th cycle samples were incubated at 72 °C for 5 min. Following PCR amplification the product was purified using High Pure PCR Purification Kit (Roche, Mannheim, Germany). The purified product was used as a template in microsequencing/SNaPshot (Applied Biosystems, Foster City, CA, USA) reaction. Conditions for the second amplification were: 96 °C for 10 s, 50 °C for 10 s, 60 °C for 30 s; cycle number: 25. The concentrations of microsequencing primers – designed adjacent to mutations – were 0.08, 0.30, 0.08 and 0.50 μM. After shrimp alkaline phosphatase treatment (in order to alter the electrophoretic mobility of the unincorporated ddNTPs), the fluorescently labelled dideoxy-terminated fragments were analysed on ABIPrism310 Genetic Analyser. Genotype assignment was automatically accomplished by Genotyper and Excel programme.

Results Voluntary PrP genotyping was started in 2003 and continued in 2004 to obtain frequency information for the commercial and indigenous breeds (Table 1 first column and Table 2). Among the commercial breeds (Table 1, first column), the highest ARR allele frequency (69.81) was obtained in the Ile de France breed, and the lowest value (19.84) in the Merino Landschaf population. The VRQ allele is present in all breeds but in the Lacaune and British Milksheep population, in some cases at a rather high frequency: Charollais 10.85%, Texel 8.72%, Ile de France 6.60%. The ARQ allele shows the highest frequency in the Merino Landschaf (73.02%) population and the lowest value in the Ile de France animals sampled (22.64%). In the four indigenous breeds (Table 2) ARQ is the most common allele (42.50 to 70.29%), ARH and VRQ are absent in some breeds, and only three al-

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Table 1 Genotype and allele frequencies in the breeds included in the Hungarian national scrapie control programme (number of sheep genotyped = 8937)

Genotype

Hungarian Merino Allele 2003–2004 2005 2006 frequencies

German Mutton Merino

Merino Landschaf

German Blackheaded

Suffolk

2003–2004

2005

2006

2003–2004

2005

2006

2003–2004

2005

2006

2003–2004

2005

2006

1207

943

312

746

1039

63

290

293

65

164

187

81

421

483

NSP1 ARR/ARR

21.11

22.87

23.54

16.03

24.40

24.35

4.76

8.28

8.53

21.54

34.15

34.22

22.22

25.18

27.54

NSP2 AHQ/AHQ AHQ/ARR

7.33

0.08 6.96

0.32 6.68

0.32 18.59

0.67 12.33

0.87 17.23

1.59

3.10

0.34 13.31

3.08

0.61

2.14

1.23 3.70

0.24 7.13

0.21 4.97

NSP3 AHQ/ARH AHQ/ARQ ARR/ARH ARR/ARQ

1.78 0.67 40.89

3.23 0.99 48.14

0.32 4.45 1.27 44.96

6.09 43.91

7.24 0.40 43.43

10.20 0.38 35.32

7.94 1.59 26.98

4.94 2.47 44.44

2.85 0.48 48.22

3.52 4.14 44.72

NSP4 ARH/ARH ARQ/ARQ ARH/ARQ ARR/VRQ AHQ/VRQ

24.22 0.44 2.89

14.17 1.16 1.82 0.08

14.42 0.64 1.48 0.11

13.14 1.60 0.32

9.65 0.27 0.94

9.53

55.56

13.30 0.71 1.66

10.56 2.48 1.04 0.21

NSP5 ARH/VRQ ARQ/VRQ VRQ/VRQ

0.67

0.50

0.11 1.70

0.24

0.62

Hungarian Merino Allele frequencies 2n

ARR AHQ ARH ARQ VRQ

0.13 0.54

0.96 0.10

0.69 3.79 39.66 41.38 0.34 1.03

6.48 1.02 44.37 24.91 1.02

0.53 55.38

52.44

50.80

18.46

6.71

1.54

6.10

10.70 0.53 1.07

0.00 19.75 1.23

1.59 1.06

1.72

German Mutton Merino

Merino Landschaf

German Blackheaded

Suffolk

2003–2004

2005

2006

2003–2004

2005

2006

2003–2004

2005

2006

2003–2004

2005

2006

2003–2004

2005

2006

900

2414

1886

624

1492

2078

126

580

586

130

328

374

162

842

966

47.00 4.56 0.56 46.11 1.78

51.82 5.22 1.08 40.68 1.20

50.74 6.10 1.17 40.30 1.70

47.44 12.66 0.80 38.94 0.16

52.95 10.46 0.40 35.39 0.80

51.30 14.63 0.19 32.82 1.06

19.84 4.76 1.59 73.02 0.79

30.17 3.79 0.52 64.14 1.38

37.88 10.24 1.02 50.85

51.54 1.54 0.00 46.15 0.77

63.72 0.30

61.23 1.34 0.27 36.63 0.53

48.15 5.56 1.23 44.44 0.62

53.92 5.23 0.59 39.31 0.95

54.97 4.55 3.31 36.23 0.93

32.93 3.05

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Table 1 continued Genotype and allele frequencies in the breeds included in the Hungarian national scrapie control programme (number of sheep genotyped = 8937)

Allele 2003–2004 frequencies

Ile de France

Charollais

Lacaune

British Milksheep

2006

2003–2004

2005

2006

2003–2004

2005

2006

2003–2004

2005

2006

2003–2004

2005

2006

109

70

113

53

384

354

129

61

66

121

260

267

72

45

89

NSP1 ARR/ARR

10.09

21.43

22.12

52.83

48.44

51.41

12.40

27.87

10.61

19.83

25.77

36.70

9.72

4.44

7.87

NSP2 AHQ/AHQ AHQ/ARR

0.92 0.92

4.29

6.19

1.89

2.34

1.41

4.13

4.23

7.12

16.67 29.17

8.89 42.22

15.73 23.60

NSP3 AHQ/ARH AHQ/ARQ ARR/ARH ARR/ARQ

0.92 0.92 17.43 26.61

0.88 6.19 36.28

3.85

15.56

26.97

27.68

51.15

1.12 0.37 45.32

13.89

24.53

0.26 0.78 32.55

1.41

14.29 32.86

20.83

13.33

20.22

NSP4 ARH/ARH ARQ/ARQ ARH/ARQ ARR/VRQ AHQ/VRQ

2.75 6.42 16.51 9.17 0.00

10.00 10.00 4.29

8.85 7.96 7.08

7.55

2.08

1.98

13.33 2.22

5.62

7.55

11.72

13.84 0.28

NSP5 ARH/VRQ ARQ/VRQ VRQ/VRQ

0.92 5.50 0.92

2.86

4.42

5.66

0.78 1.04

Texel Allele frequencies 2n

ARR AHQ ARH ARQ VRQ

1.64 3.28 1.64

1.52

31.15

37.88

3.31 1.65 43.80

25.58 0.00 10.08

13.11

24.24

24.79

13.08

8.24

6.94

9.84

9.09

2.48

1.54 0.38

1.12

1.39 1.39

1.69 0.28

8.53 1.55

6.56 4.92

16.67

1.55 40.31

Ile de France

Charollais

Lacaune

British Milksheep

2003–2004

2005

2006

2003–2004

2005

2006

2003–2004

2005

2006

2003–2004

2005

2006

2003–2004

2005

2006

218

140

226

106

768

708

258

122

132

242

520

534

144

90

178

37.16 2.29 20.64 31.19 8.72

49.29 2.14 12.14 32.86 3.57

50.00 3.54 7.08 33.63 5.75

69.81 0.94 0.00 22.64 6.60

72.14 1.30 0.39 18.88 7.29

72.88 1.55

38.37 0.00 0.78 50.00 10.85

50.00 4.10

34.09 0.76

54.23 4.23

52.27 12.88

63.67 4.12 0.19 31.46 0.56

35.42 38.89 0.00 24.31 1.39

32.22 37.78 1.11 28.89

29.78 41.01

32.79 13.11

45.87 3.72 0.83 48.35 1.24

17.37 8.19

40.58 0.96

29.21

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leles are present in the Cikta population. With 14 genotypes present the Hortobágy Racka is the most polymorphic breed, the other three have 5 to 6 genotypes only. These differences can be explained by the larger population size of the former breed. The accepted breeding programme modifications had been introduced by 1st of April, 2005 in the most important commercial breeds. The genotyping results for the years 2005 and 2006 are presented in Table 1 (2nd and 3rd columns, respectively). Table 2 Genotype and allele frequencies in the indigenous sheep breeds of Hungary (Fésüs et al., 2004) (number of sheep genotyped = 330) Genotype

Allele frequencies

NSP1

ARR/ARR

NSP2

AHQ/AHQ AHQ/ARR

NSP3

NSP4

NSP5

Gyimes Racka

Hortobágy Racka

Tsigaja

Cikta

57

140

64

69

5.26

5.00

3.13

1.45

4.29 6.43

6.25

4.35

AHQ/ARH AHQ/ARQ ARR/ARH ARR/ARQ ARH/ARH ARQ/ARQ ARH/ARQ ARR/VRQ AHQ/VRQ

1.75 42.11 47.37 1.75

ARH/VRQ ARQ/VRQ VRQ/VRQ

1.75

ARR AHQ ARH ARQ VRQ

27.19 0.88 70.18 1.75

7.14 12.14 4.29 25.00 0.71 17.86 10.00 2.14 1.43

14.49 45.31

33.33

40.63 3.13

46.38

1.43 2.14

1.56

23.93 17.86 12.14 42.50 3.57

28.91 3.13 1.56 65.63 0.78

20.29 9.42 70.29

Discussion According to the accepted scrapie genotyping breeding programme all breeding rams should be genotyped, and only NSP1, NSP2 and NSP2 rams are allowed for breeding. When possible, avoiding the use of ARR/ARQ (NSP3) rams is highly recommended. Ewes carrying at least 1 VRQ allele may be moved Acta Veterinaria Hungarica 56, 2008

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from the holding solely for slaughter. In the breeds where VRQ and/or ARQ allele frequencies are high (Charollais, Ile de France, Texel), mating of NSP1 and NSP2 group animals is strongly advised to improve the efficiency of the selection programme. The indigenous breeds are exempted from the programme. When compared to the 2003 and 2004 data, the frequency of the ARR allele increased in all breeds but in the Charollais and British Milksheep population. The selection aimed at increasing the ARR allele frequency seems to be more efficient in breeds of smaller population size, such as the Merino Landschaf, German Blackheaded, Texel and Lacaune. The VRQ allele has disappeared in two breeds (Merino Landschaf and British Milksheep), its frequency decreased in the Hungarian Merino, German Blackheaded, Texel and Lacaune breeds, and increased in some others (German Mutton Merino, Suffolk, Ile de France, Charollais). The ARQ allele frequency has decreased in most breeds, first all in those which are represented in rather small numbers in Hungary, and slightly decreased in the Texel, Charollais and British Milksheep population. Notable allele frequency changes in the individual breeds included in the Hungarian breeding programme will be observed only after a few more generations. The efficiency of the genotyping programme will mostly depend on the number of genotyped individuals in each breed and also on the inclusion of the female population in the programme. Finally we have to mention that recently so-called ‘atypical’ scrapie cases have been observed in some European countries (Benestad et al., 2003; Buschmann et al., 2004; De Bosschere et al., 2004; Lühken et al., 2004; Onnasch et al., 2004; Orge et al., 2004). This will prompt the European Union to re-evaluate the existing compulsory breeding programme, and some probable modifications can be expected in the near future. References Áldásy, P. and Süveges, T. (1964): Incidence of scrapie in the domestic sheep population (in Hungarian, with English abstract). Magyar Állatorvosok Lapja 19, 463–466. Belt, P., Muilemann, I., Schreuder, B., Bos-De-Ruijter, J., Gielkens, A. and Smits, M. (1995): Identification of five allelic variants of the sheep PrP gene and their association with natural scrapie. J. Gen. Virol. 76, 509–517. Benestad, S. L., Sarradin, P., Thu, P., Schönheit, J., Tranulis, M. A. and Bratberg, B. (2003): Cases of scrapie with unusual features in Norway and designation of a new type, Nor98. Vet. Rec. 153, 202–208. Buschmann, A., Biacabe, A.-G., Ziegler, U., Bencsik, A., Madec, J.-Y., Erhardt, G., Lühken, G., Baron, T. and Groschup, M. H. (2004): Atypical scrapie cases in Germany and France are identified by discrepant reaction patterns in BSE rapid tests. J. Virol. Methods 117, 27–36. Butler, D. (1998): Doubts over ability to monitor risk of BSE spread to sheep. Nature 395, 6–7. De Bosschere, H., Roles, S., Benestad, S. L. and Vanopdenbosch, E. (2004): Scrapie case similar to Nor98 diagnosed in Belgium via active surveillance. Vet. Rec. 155, 707–708. DEFRA (2004): The National Scrapie Plan for Great Britain. www.defra.gov.uk/nsp. Accessed October 2004.

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European Commission (2003): Commission Decision of 13 February 2003 Laying Down Minimum Requirements for the Establishment of Breeding Programmes for Resistance to Transmissible Spongiform Encephalopathies in Sheep (2003/100/EC). Fésüs, L., Zsolnai, A., Horogh, G. P. and Anton, I. (2004): Scrapie in sheep. 2. Prion genotype frequencies in the domestic indigenous breeds (in Hungarian, with English abstract). Magyar Állatorvosok Lapja 126, 670–675. Foster, J. D., Hope, J. and Fraser, H. (1993): Transmission of bovine spongiform encephalopathy to sheep and goats. Vet. Rec. 133, 339–341. Houston, F., Goldmann, W., Chong, A., Jeffrey, M., González, L., Foster, J., Parnham, D. and Hunter, N. (2003): BSE in sheep bred for resistance to infection. Nature 423, 498. Hunter, N., Goldmann, W., Smith, G. and Hope, J. (1994): The association of a codon 136 PrP gene variant with the occurrence of natural scrapie. Arch. Virol. 137, 171–177. Hunter, N., Poster, J. and Hope, J. (1992): Natural scrapie in British sheep: breeds, ages and PrP gene polymorphisms. Vet. Rec. 130, 389–392. Jeffrey, M., Ryder, S., Martin, S., Hawkins, S. A. C., Terry, L., Berthelin-Baker, C. and Bellworthy, S. J. (2001): Oral inoculation of sheep with the agent of bovine spongiform encephalopathy (BSE). 1. Onset and distribution of disease-specific PrP accumulation in brain and viscera. J. Comp. Pathol. 124, 280–289. Lühken, G., Buschmann, A., Groschup, M. H. and Erhardt, G. (2004): Prion protein allele A136H154Q171 is associated with high susceptibility to scrapie in purebred and crossbred German Merinoland sheep. Arch. Virol. 149, 1571–1580. Onnasch, H., Gunn, H. M., Bradshaw, B. J., Benestad, S. L. and Bassett, H. F. (2004): Two Irish cases of scrapie resembling Nor98. Vet. Rec. 155, 636–637. Orge, L., Galo, A., Machado, C., Lima, C., Ochoa, C., Silva, J., Ramos, M. and Simas, J. P. (2004): Identification of putative atypical scrapie in sheep in Portugal. J. Gen. Virol. 85, 3487–3491.

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