Genetic predisposition of some Bulgarian sheep breedsto the scrapie disease

Ivo Sirakov • Raiko Peshev • Lilia Christova

Received: 30 August 2010 / Accepted: 15 April 2011 / Published online: 30 April 2011

� Springer Science+Business Media, LLC 2011

Abstract The aim of this study is to investigate the

profile of ovine PrP gene by amino acid polymorphism at

codons 136, 141, 154, and 171 for determining the genetic

predisposition to the Scrapie disease for the tribal sheep

and rams, with different numbers and distribution in

Bulgaria. Three hundred twenty four animals originating

from 41 tribal herds comprising eight breeds were included

in the study. DNA was isolated from blood samples spe-

cifically amplified by PCR and sequenced. The alignments

of codons 136, 141, 154, and 171 were determined. Based

on the sequencing, it was established that Bulgarian breeds

belong to the second and third risk groups, those with low

and moderate risk of Scrapie disease. Establishment of 11

genotypes in Synthetic Population Bulgarian Milk breed

reveals it to have the highest risk of the Scrapie disease;

moreover, the conducting of the program will be more

difficult in comparison with other investigated breeds.

Evidence for the internal cross breeding is the presence of

the five or six genotypes in the Copper-Red Shoumen,

Replian, Karakachan, and Duben Bulgarian native breeds.

Keywords PrP gene � Scrapie disease � PCR � Genotypes

Introduction

Scrapie is a neural degenerative and lethal disease with a

long incubation period, affecting the central nervous sys-

tem of sheeps and goats. It has been known in Europe for

more than 250 years [1]. The etiologic agent is still not

fully characterized and there are unexplained facts

regarding the pathogenesis of the disease. The disease is

associated with the progressive accumulation of patholog-

ical PrPSc isoforms, which is derived from the normal

cellular PrPC [2, 3]. By oral transmission, the agent enters

the intestines, where by M-cells [4] or dendritic cells [5] it

passes the mucous barrier. Its accumulation is in the tan-

gible body macrophages, and later in the follicular den-

dritic cells of the ileal Peyer’s patch. Through the ferritin

pathway it affects the submucosal plexus [6]. Dissemina-

tion of PrPSc is via a lymph-hematogenen path in the

lymphoreticular system. Through the migration of the

autonomous nervous system [7–9] it affects the central

nervous system. Entry through conjunctiva is also possible

[10], as well as by means of scarification [11]. In the herds,

dissemination of PrPSc takes place through the placenta

during labor [5]. Furthermore, the prion agent has been

demonstrated to be present in milk and colostrum [12, 13].

The development of the disease is determined by the

genetic susceptibility of the sheep, and contact with the

infectious agent [14, 15]. The gene encoded prion protein is

localized in the 13th chromosome [16], with a size of

31 kb. It contains two non-coding and one coding exons

with an open reading frame spanning 236 codons [17].

More than 25 polymorphic codons with 40 different hap-

lotypes, derived from 32 amino acid substitutions of the

ovine prion gene have been described [18]. Polymorphism

has been detected at 29 different codons. Polymorphism at

codon 141 (L/F) is responsible for the manifestation of the

I. Sirakov (&) � R. Peshev

National Diagnostic and Research Veterinary Medical Institute,

Prof.Dr ‘‘G.Pavlov’’, 15 ‘‘P. Slaveykov’’ Blvd, Sofia, Bulgaria

e-mail: insir_2000@yahoo.co.uk

L. Christova

Institute of Biophysics, Bulgarian Academy of Sciences,

Acad. G. Bontchev Str. Bl. 23, Sofia, Bulgaria

123

Virus Genes (2011) 43:153–159

DOI 10.1007/s11262-011-0615-7

atypical scrapie [19, 20]. The sheep’s resistance to the

classical scrapie type is predetermined by three amino acid

positions: 136 (A/V), 154 (R/H), and 171 (Q/R/H) [21–23].

Sheep, homozygous for VV at codon 136 and for QQ at

171 are observed to have a shorter incubation period, while

the VA heterozygous at codon 136 and for RQ at codon

171—an extended incubation period for the same disease

[23, 24]. On the other hand, the homozygous codons

136-AA and 171-RR, and the heterozygous codon 171-QR

are associated with resistance to the classical Scrapie [23].

Sheep, homozygous for ARR allele are resistant to the

disease [14], while among the heterozygous sheep with the

same allele the disease is rare [26]. Meanwhile the resis-

tance to the Scrapie disease conditioned by the homozy-

gous ARR allele does not protect the sheep against the

Bovine Spongiform Encephalopathy (BSE) [27, 28]. Due

to its unconventional appearance, the eradication of the

scrapie disease is very difficult [29] and requires clear

determination of sheep’s genotype and their expected

susceptibility.

The clear necessity for genomic typifying is emphasized

by the fact, that on the ground of its results the under-

standing of the disease will improve and in turn be of great

importance to the breeding programs aimed at eradicating

the disease in livestock. The relationship between the

scrapie causative agent and the BSE, as well as the zoo-

notic new variant of Creutzfeldt-Jakob disease in humans

gives us strong reasons to continue with the researches for

the restriction of Scrapie disease spreading.

The main aim of this study was to investigate the genetic

predisposition of tribal flocks of several indigenous sheep

breeds with different populations and distribution, in many

regions of Bulgaria, to the Scrapie disease. For this purpose,

we explored the profile of the ovine PrP gene through the

amino acid polymorphism at codons 136, 141, 154, and 171.

Materials and methods

Blood samples from 324 animals (170 rams and 154 sheep)

were tested. The animals originated from 41 native tribal

flocks, with population sizes of more than 10,000, con-

sisting of the following sheep breeds: Copper-Red Shou-

men sheep (n = 47); Synthetic population Bulgarian milk

SPBM (n = 49); Blackhead Pleven breed (n = 40); Stara

Planina Tzigay (n = 37); population size over 5000—

Replyan sheep (n = 39); Karakachan sheep (n = 31); and

Local Stara Zagora sheep (n = 40); population size under

5000—Duben sheep (n = 41).

For DNA isolation, we used the commercial kit Illustra

blood genomic Prep Mini Spin Kit (GE Healthcare, UK),

closely following the kit instructions. The genomic DNA

was amplified by means of PCR, using the following pairs

of primers: forward (G30) 50-CATTTGATGCTGACACC

CTCTTTA-30 and reverse (G16) 50-ATGAGACACCA

CCACTACAGGGCT-30. The reaction was executed at

25 ll volume, using Illustra puReTaq Ready-To-Go PCR

Beads (GE Healthcare, UK). The following procedure was

accomplished via Thermocycler QB-96 (LKB): denatur-

ation at 95�C for 10 min, followed by 40 cycles at 95�C for

20 s, 60�C for 30 s, and 72�C for 3 min, with a final

extension step of 72�C for 10 min. The obtained DNA

extraction and the PCR products were electrophoresed on a

2% agarose gel (USB Corporation, Cleveland, OH, USA)

with Ethidium bromide (1 mg/ml) and visualized with UV

transilluminator. After purification of the PCR products

with S400 columns (GE Healthcare, UK), the sequencing

PCR reactions by a Thermocycler QB-96 (LKB) were

applied. The amount of primers used for the sequencing of

the PCR products was 5 pmol/ll. The following sequenc-

ing primers were applied: forward (SWF3) 50-GTAAGC

CAAAAACCAACATGAAGC-30 and reverse (SWR6.2)

50-TCGCTCCATTATCTTGATGTCAGTTT-30. The prod-

uct used for the DNA sequencing was the DYEnamic ET

Dye Terminator Cycle Sequencing Kit. PCR sequencing

was executed as follows: 25 cycles at 95�C for 30 s, 54�C

for 30 s, and 72�C for 1 min. The PCR products were

purified by G50 columns or ethanol precipitation tech-

nique, according to the manufacturer’s instructions (GE

Healthcare, UK). The PCR sequencing products were

checked by the sequencing machine—MegaBace 1000

(Amersham Biosciences). The polymorphisms of the fol-

lowing codons: 136, 141, 154, and 171 were defined by the

sequencing peaks. Data analysis was carried out with

MEGA4 computer software [30]. Genotypic (fij) and allelic

(pi) frequencies were calculated with a formulae, used by

Gama et al. [31]: fij ¼ nij=N and pi ¼ ð2fii þ R fijÞ=2,

where nij is the number of animals with the ij genotype; fijand fii are heterozygous and homozygous genotype fre-

quencies, respectively; pi is allelic frequency; and N is the

total number of animals. The allelic frequencies within and

between investigated breeds were compared by 2 9 2

contingency tables with v2 test set, at 95% confidence

interval and critical probability of 0.05.

Results

The amount of the DNA was ranged up 3–5 lg/ml with

purity up 1.3–1.74. Five allelic variants of the PrP gene at

136, 154, and 171 codons (ARR, ARQ, ARH, AHQ, and

VRQ) were observed as the ARR and ARQ alleles were the

most frequent in the studied breeds.

154 Virus Genes (2011) 43:153–159

123

The ARQ allelic frequency in Copper-Red Shoumen

(63.82%), Replyan (64.09%), and Stara Planina Tzigay

(59.74%), breeds was significantly higher than in Local Stara

Zagora (v2 = 7.0, P = 0.0081; v2 = 7.47, P = 0.0063;

v2 = 6.47, P = 0.011, respectively). The ARQ frequency of

Replyan breed was significantly higher than that of Duben

(45.05%) sheep (v2 = 4.09, P = 0.0432). Among the other

breeds, there was no considerable difference found in the

ARQ allelic occurrence (P [ 0.05): Blackhead Pleven breed

(53.75%), SPBM (49.95%), Karakachan sheep (48.4%), and

Duben sheep (45.05%) (Fig. 1).

The ARR allele rate was significantly higher in Karaka-

chan (46.8%), Duben (43.9%), and Local Stara Zagora

(41.25%) sheep (v2 = 9.78, P = 0.0018; v2 = 7.24,

P = 0.0071; v2 = 6.73, P = 0.0095, respectively) com-

pared to the Replyan sheep (21.80%). In the other breeds was

not found a significant difference (P [ 0.05) of the ARR

allele frequency. It ranged from 26.53% (SPBM) to 41.25%

(Local Stara Zagora sheep). A significant difference in fre-

quency distribution between the ARR and ARQ alleles

was found in Copper-Red Shoumen, SPBM, Blackhead

Pleven breed, Stara Planina Tzigay, and Replyan sheep

(v2 = 13.91, P = 0.0002; v2 = 9.44, P = 0.0021; v2 =

5.49, P = 0.0191; v2 = 8.88, P = 0.0029; v2 = 23.61,

P = 0.0000, respectively). In the other breeds, there was no

relevant difference found between the distribution frequen-

cies of those two alleles (Fig. 1). AHQ, ARH, and VRQ

alleles have the lowest rate of spread. The AHQ allele was

not detected only in animals from Local Stara Zagora breed.

Its frequency was highest at SPBM (16.01%) compared

to the Copper-Red Shoumen (2.13%), Blackhead Pleven

breed (5%), and Karakachan breeds (4.8%) (v2 = 9.03,

P = 0.0027, v2 = 3.89, P = 0.0485; v2 = 5.02, P =

0.025, respectively). The AHQ allele spread among the other

breeds did not manifest a statistically significant difference

(P [ 0.05) and ranged from 2.1% (Copper-Red Shoumen) to

12.82% (Replyan sheep). The AHQ allelic frequency was

significantly lower (P \ 0.05) than those of the ARR allele

in five breeds (Copper-Red Shoumen: v2 = 24.03, P =

0.0000; Blackhead Pleven breed: v2 = 16.81, P = 0.0000;

Stara Planina Tzigay: v2 = 14.19, P = 0.002, Karakachan

sheep: v2 = 24.73, P = 0.0000; and Duben sheep:

v2 = 20.50, P = 0.0000). No relevant differences were

found in the frequency of occurrence of AHQ allele in the

other two breeds, SPBM and Replyan breeds. Frequency of

occurrence of the AHQ allele was significantly lower

(P \ 0.05) than that of the ARQ allele for all investigated

seven breeds (Fig. 1). The ARH allele was detected in the

three investigated Bulgarian breeds; SPBM (6.12%), Stara

Planina Tzigay (4.05%), and Blackhead Pleven breed

(1.25%). As the allele frequency was significantly lower

(P \ 0.05) than those of the ARR (v2 = 13.3, P = 0.0003;

v2 = 22.2, P = 0.0000; v2 = 25.08, P = 0.0000) and ARQ

(v2 = 40.3, P = 0.0000; v2 = 49.55, P = 0.0000; v2 =

46.88, P = 0.0000). The ARH allelic frequency was sig-

nificantly lower (v2 = 4.36, P = 0.0369) than those of AHQ

for the SPBM breed only (Fig. 1). The sheep with VRQ allele

is highly sensitive to the Scrapie disease. In the research, we

found VRQ allele in all investigated breeds with the excep-

tion of the Karakachan sheep. Its frequency of occurrence

was significantly higher (P \ 0.05) in Local Stara Zagora

sheep (21.25%) than in the other breeds like Duben sheep

(3.65%), SPBM (3.06%), Stara Planina Tzigay (2.70%),

Copper-Red Shoumen (2.13%), and Replyan sheep (1.28%)

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1 42 3 5 6 7 8

Allele ARR Allele ARQ Allele AHQ Allele ARH Allele VRQ

#**

#^ ^

!

?

Fig. 1 Genotype frequency of five allelic variants (ARR, ARQ,

ARH, AHQ, and VRQ) of the PrP gene for 136, 154, and 171 codons

in investigated breeds: Copper-Red Shoumen sheep (1), Synthetic

population Bulgarian Milk (2), Blackhead Pleven breed (3), Stara

Planina Tzigay (4), Replyan sheep (5), Karakachan sheep (6), Duben

sheep (7), and Local Stara Zagora sheep (8). Note: significant

differences of allelic frequency between investigated breeds are

marked by * for ARR, # for ARQ, ^ for AHQ, ! for ARH, and ? for

VRQ alleles

Virus Genes (2011) 43:153–159 155

123

(Fig. 1). The frequency of occurrence of the VRQ allele was

significantly lower than those of ARR and ARQ alleles for 7

of the investigated breeds except for the Local Stara Zagora

breed. In two breeds (SPBM and Replyan sheep), the VRQ

distribution was significantly lower than AHQ (v2 = 7.47,

P = 0.0063; v2 = 17.34, P = 0.0067, respectively). In the

five investigated breeds, ARH allele was not observed, but in

the other three breeds no difference was established between

VRQ and ARH allelic frequency.

Based on these five allele variants, 12 scrapie risk

genotypic groups were specified, according to the classifi-

cation of [20]: ARR/ARR, ARR/ARQ, ARR/AHQ, ARR/

ARH, AHQ/AHQ, AHQ/ARH, AHQ/ARQ ARH/ARQ,

ARQ/ARQ, ARR/VRQ, ARQ/VRQ, and VRQ/VRQ

(Table 1).

The ARR/ARR genotype, determining the lowest level

of risk for infection with Scrapie was found in all studied

breeds and the difference of its frequency of occurrence

was insignificant. ARR/ARH genotype was not found in

four breeds (Copper-Red Shoumen, Replyan, Karakachan,

and Local Stara Zagora sheep). In the other four breeds,

significant difference was not established. The frequency of

occurrence of genotype ARR/ARQ of Karakachan sheep

was significantly higher compared to SPBM (v2 = 5.82,

P = 0.0158), Replyan sheep (v2 = 4.91, P = 0.0267), and

Local Stara Zagora sheep (v2 = 5.12, P = 0.0236). The

ARR/AHQ genotype was found in SPBM breed (2.04%)

only (Table 1). No significant difference was found

between the ARR/ARH and ARR/ARR, ARR/AHQ, AHQ/

AHQ, AHQ/ARH, ARR/VRQ, ARQ/VRQ genome type.

Significant differences were established between ARR/

ARQ (v2 = 9.5, P = 0.0021), AHQ/ARQ (v2 = 6.0,

P = 0.0144) and ARQ/ARQ (v2 = 12.0, P = 0.0005). The

AHQ/AHQ genotype was found in three breeds with

insignificant differences in its frequency of occurrence:

SPBM (4.08%), Replyan (2.56%), and Karakachan sheep

(3.23%). The AHQ/ARH genotype was only observed in

SPBM breed (2.04%). The AHQ/ARQ was presented

in seven breeds as its frequency of occurrence was highest

in the Replyan sheep (20.51%) and was significantly dif-

ferent compared to Copper-Red Shoumen (v2 = 5.48,

P = 0.0192) and Karakachan sheep (v2 = 4.61, P =

0.0319). The ARH/ARQ genotype was observed only in

three of the investigated breeds (SPBM, Blackhead Pleven,

Table 1 Frequency distribution of different PrP gene types at codons 136, 154, and 171 and the risk groups to which they belong

Genotype Risk

group

Breeds

Copper-Red

Shoumen

Synthetic population

Bulgarian Milk

Blackhead

Pleven breed

Stara Planina

Tzigay

Replyan

sheep

Karakachan

sheep

Duben

sheep

Local Stara

Zagora

(n = 47) (n = 49) (n = 40) (n = 37) (n = 39) (n = 31) (n = 41) (n = 40)

ARR/

ARR

1 0.1489 0.1020 0.1000 0.1081 0.1026 0.2258 0.2439 0.2000

ARR/

ARQ

2 0.3404 0.2245 0.3000 0.3514 0.2308 0.4838 0.3171 0.3000

ARR/

ARH

2 0 0.0408 0.0250 0.0540 0 0 0.0732 0

ARR/

AHQ

2 0 0.0204 0 0 0 0 0 0

AHQ/

AHQ

3 0 0.0408 0 0 0.0256 0.0323 0 0

AHQ/

ARH

3 0 0.0204 0 0 0 0 0 0

AHQ/

ARQ

3 0.0426 0.1633 0.0750 0.0540 0.2051 0.0323 0.0732 0

ARH/

ARQ

3 0 0.0612 0.0250 0.0270 0 0 0 0

ARQ/

ARQ

3 0.4255 0.2653 0.3000 0.3514 0.4103 0.2258 0.2195 0.1500

ARR/

VRQ

4 0 0.0408 0.1 0 0 0 0 0.1250

ARQ/

VRQ

5 0.0426 0.0204 0.0750 0.0540 0.0256 0 0.0732 0.1500

VRQ/

VRQ

5 0 0 0 0 0 0 0 0.0750

Note: All tested samples were LL homozygous for the 141 codon

156 Virus Genes (2011) 43:153–159

123

and Stara Planina Tzigay) and its frequency of occurrence

were insignificant. The most common genotype ARQ/ARQ

was with a significantly higher percent of distribution in

Copper-Red Shoumen and Replyan breeds, than the Duben

(v2 = 4.21, P = 0.0403 and v2 = 4.35, P = 0.0421) and

Local Stara Zagora (v2 = 7.83, P = 0.051 and v2 = 6.66,

P = 0.0099) breeds. The ARR/VRQ genotype was found in

three breeds and no difference in its percentage of distribu-

tion was established. The ARQ/VRQ genotypes were found

in seven breeds with low frequency (Table 1).

The highly associated with the Scrapie infection sus-

ceptibility VRQ/VRQ genotype, was detected only in

Local Stara Zagora sheep with a very low percent of dis-

tribution (7.5%).

The genotypes ARR/ARR, ARR/ARQ and ARQ/ARQ

were predominant among the Bulgarian breeds with fre-

quency of occurrence of 15.1, 29.6, and 31.2%, while

VRQ/VRQ genotype occurred in three of the 324 tested

sheep with a lower frequency of occurrence of 0.9%. The

haplotype variant ARH was found in seven of the 324

tested sheep only.

Discussion

Management of the disease is associated with risk assess-

ment [32], which depends on the knowledge regarding the

etiology and pathogenesis, the mechanisms of the agent’s

transmission, the spread of the disease and genetic factors.

There is no literature data on the application of medicines

against Scrapie in sheep and goats. The polyene antibiotic

MS-8209 protects the experimentally infected immunode-

ficient SCID mice against infection with Scrapie [33]. The

lentivector-mediated RNAi suppresses the accumulation of

PrPSc in scrapie-infected neuronal cells [34]. Because of

this, the selection of disease resistant animals is of vital

importance for the fight against the Scrapie disease. The

management of Scrapie is described in the European

Community by the EU Directive 999/2001. Furthermore,

the EU Commission in the 2003/100/EU decision lists the

conditions and rules for implementation of breeding pro-

grams, as part of the management of Scrapie. This docu-

ment requires each country to prepare its own breeding

program for the herds of high genetic merit, in order to

increase the frequency of ARR allele and to reduce the

alleles contributing to the susceptibility of the disease. The

low frequence in occurrence of the ARR and the use of a

small number of sires carrying this allele hides a risk of

increasing the percentage of inbreeding and the possibility

of an antigenic drift [35]. In this case, it is suitable to focus

on the elimination of the most sensitive alleles in the breed

[36]. Furthermore, the effectiveness of selection, for

increased distribution, of a particular allele depends on its

initial distribution [37].

Dawson et al., [35, 38], defined the significance of the

existing polymorphism at codons 136, 154, and 171 and its

close connection to the risk of Scrapie infection. They

specified 15 genome types and five risk groups. In the

investigated Bulgarian sheep, we determined 12 basic

genome types, belonging to the second and third risk

groups with low and middle sensitivity to the Scrapie

disease. This is the reason for the minor susceptibility and

low frequency of Scrapie disease among Bulgarian sheep.

This finding is being confirmed by the small percentage of

positive Scrapie diagnoses (seven out of 36,066 for the

2008—August 2010 time frame).

The observed high conservativity of the 136 codon,

represented by the AA homozygosity and low percent of

QQ homozygosity at codon 171 was found only in the

Karakachan sheep. Taking into consideration the statement

of O’Rourke et al. [25] that AA homozygosity at codon

136, as well as, the RR homozygosity and RQ heterozy-

gosity at codon 171 are associated with the resistance to the

clinical manifestation of Scrapie, we found this breed to be

the most resistant. The AV heterozygosity, observed in all

other breeds along with the QQ heterozygosity at codon

171, determines the different levels of risk for the devel-

opment of clinical symptoms among these breeds.

No polymorphism was found at codon 141 similar to

Thorgeirsdottir et al. [39]. The most probable reason for the

lack of polymorphism at codon 141 is the collection of

samples from different regions and farms in Bulgaria.

Regarding the role of polymorphism at the codon 154,

the situation is not fully clear yet. The histidine, according

to Dawson et al. [38] and Thorgeirsdottir et al. [39] is

connected to a high resistance and a prolonged incubation

period of the disease. However, Ekateriniadou et al. [40]

discovered increased sensitivity even in the absence of V at

the codon 136 in Chios breed, which illustrates the existing

connection between the agent’s strains and the sheep’s

breed [38]. In Romanov, Suffolk and the Finn Dorset sheep

the H allele at codon 154 is present in both clinically sick

and healthy animals [15, 38, 41]. On the other hand, in

Cheviot sheep and Texel breed the AHQ allelic variant

seems to be associated with resistance to Scrapie [24, 42].

We found the AHQ allelic variant in all investigated breeds

except the Local Stara Zagora sheep. Specified as a ‘‘wild

type’’ ARQ allele [43] is associated with susceptibility to

Scrapie [21, 44–46], especially in sheep flocks with rare

predominant or without VRQ allele [47]. The VRQ allele is

a target for some Scrapie strains, while others prefer the

ARQ allele [42]. In the rare Karakachan, Duben and Local

Stara Zagora sheep the ARR allele frequency was the

highest. It is very likely that this is a result of the synergism

between two factors: the unplanned breeding of animals

Virus Genes (2011) 43:153–159 157

123

with ARR allele in the parent flocks and the small popu-

lations of the breeds. That is why, in these breeds and

Replyan sheep it is appropriate to apply the Dutch model of

selection, which includes mild (using homozygous and

heterozygous ARR rams for breeding, without prefer-

ences), moderate (using preferentially homozygous ARR

rams) and severe selection (using only homozygous ARR

rams) [48]. The prevalence of the ARR allele in Local

Stara Zagora sheep (41.25%) allows the simultaneous

elimination of homozygous VRQ. The absence of VRQ

allele in Karakachan breed, the high percentage of ARR

haplotype and the different combinations of allelic vari-

ants, indicate that this breed is more resistant to Scrapie

than the others are. Therefore, with this breed, a direct

application of a moderate selection is possible. A possible

strategy for reducing inbreeding in these breeds is the

factorial mating, where every dam is mated to several sires

and vice versa [49, 50].

The largest number of combinations between different

allelic variants was observed in the SPBM breed (Table 1).

The most likely reason would be the multi-breeding, cross-

breeding, and purposeful selection. The considerable

genotype diversity in the above mentioned breed, along

with the absence of specialized breeding programs

increases the risk of the formation of highly sensitive to

Scrapie generations. The diagnosed Scrapie cases among

this breed and its cross breeds, as well as the absence of the

disease among the other investigated breeds confirms the

statement. This is supported by the fact that only one case

of all investigated breeds in this study, diagnosed with

Scrapie for the period of 2008–2010, was in the SPBM

breed. Having in mind that, it is possible to apply geno-

typing in the males and females of this breed, and eliminate

VRQ carriers and ARQ/ARQ rams, because this method

achieves maximum control [51]. Another option is to

increase the heterozygous threshold to 30% for the ARR

allele (in order to prevent genetic bottleneck), then to

continue the selecting only for ARR homozygous [52],

while simultaneously removing the VRQ carriers. In the

other three breeds with large populations (Copper-Red

Shoumen, Blackhead Pleven breed, and Stara Planina

Tzigay), the most economically profitable strategy associ-

ated with the smallest loss of animals can be applied. For

example, the Spanish model strategy for Merino sheep

breed—genotyping of male and removal of VRQ rams

carriers and ARQ/ARQ [51].

The PrP genotypes, determined in the tested Bulgarian

sheep breeds, belong to the second and third risk groups,

which are associated with low and moderate risk for the

development of the disease, except the SPBM breed

determined to be a breed with the highest risk of Scrapie

infection. The development of an appropriate Scrapie

resistant breeding program for this breed would be more

difficult, than for the other investigated breeds. Moreover,

as of now there is no approved selective breeding program

for Scrapie resistant flocks in Bulgaria. The results offer the

advantage of better understanding the disease and would be

useful for breeding programs seeking the eradication of the

Scrapie disease in Bulgaria.

Acknowledgment The authors would like to express their gratitude

to Prof. Zichichi from World Federation of Scientists for the gener-

ously offered financial support.

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