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1189 
Original Article 

Biosci. J., Uberlândia, v. 31, n. 4, p. 1189-1199, July/Aug. 2015 

PRION PROTEIN GENE POLYMORPHISMS AND ACCUMULATION OF 
PATHOGENIC PRION PROTEIN (PrP

Sc
) IN A HERD WITH PREVIOUSLY 

CONFIRMED SCRAPIE CASES 
 

POLIMORFISMOS DO GENE DA PROTEÍNA PRIÔNICA CELULAR (PRNP) E 
DISTRIBUIÇÃO DA PROTEÍNA PRIÔNICA ALTERADA (PrPSc) EM REBANHO 

COM SURTO ANTERIOR DE SCRAPIE 
 

Cristina Santos SOTOMAIOR
1
; David DRIEMEIER

2
; Juliano LEAL

3
;  

Rüdiger Daniel OLLHOFF
1
; Vanete THOMAZ-SOCCOL

4
 

1. Graduate Program in Animal Science, School of Agricultural Sciences and Veterinary Medicine – Pontifícia Universidade Católica do 
Paraná - PUCPR, São José dos Pinhais, Paraná, Brazil. cristina.sotomaior@pucpr.br; 2. Departamento de Patologia Clínica Veterinária, 
Faculdade de Veterinária - UFRGS, Porto Alegre, RS, Brazil; 3. Programa de Pós-Graduação em Ciências Veterinárias, Faculdade de 
Veterinária - UFRGS, Porto Alegre, RS, Brazil; 4. Programa de Pós-Graduação em Processos Biotecnológicos - UFPR, Curitiba, PR, 

Brazil. 

 
ABSTRACT: Scrapie in sheep is associated with at least three polymorphisms in the prion protein gene (PRNP) 

on codons 136, 154, and 171. Countries where scrapie is endemic have been using breeding programs based on selection 
for the most resistant alleles. There are some PRNP genotyping data on sheep in Brazil, and scrapie has sporadically been 
observed since 1978. Paraná is the Brazilian state where most of the cases of scrapie have been diagnosed. A flock that had 
three clinical scrapie cases in 2003 and 2004 was genotyped (128 sheep: 53 pure Hampshire Down and 75 crossbred) and 
slaughtered (111 sheep: 47 pure Hampshire Down and 64 crossbred) in 2006. Samples of lymphoid and central nervous 
tissues were examined by immunohistochemistry (IHC) for altered prion protein (PrPSc). Six genotypes were detected in 
the 128 genotyped animals: ARR/ARQ was the most frequent (45.3%), followed by ARQ/ARQ (28.1%), ARR/ARR 
(14.1%), and ARQ/VRQ (8.6%). ARR/VRQ and ARQ/AHQ showed less than 2.5% genotype frequency. IHC identified 
16 positive sheep. Palatine tonsil tissue had the highest percentage of reactive samples: 81.25% of the total positive 
samples. Of these 16 positive animals, nine (56.25%) had genotype ARR/ARQ, five (31.25%) had genotype ARQ/ARQ, 
and the remaining two (12.5%) had genotype ARQ/VRQ. All the positive animals were clinically healthy, and therefore 
represented 14.14% of pre-clinical cases of scrapie in this flock. 

 
KEYWORDS: Ovis aries. Prion. Immunohistochemistry. Genotyping. 

 
INTRODUCTION 

 
Scrapie is a fatal, neurodegenerative disease 

that affects sheep and goats and belongs to the 
group of transmissible spongiform encephalopathies 
(TSE) or prion diseases. In these prion diseases the 
normal cellular form of the prion protein (PrPC) 
undergoes a conformational change to the infectious 
form (PrPSc). The deposition of PrPSc in tissues of 
the central nervous system and lymphoreticular 
system characterizes the disease (PRUSINER, 
1998). 

For sheep exposed to the scrapie infectious 
agent, allelic variation at codons 136 (alanine, 
A/valine, V), 154 (histidine, H/arginine, R), and 171 
(histidine, H/glutamine, Q/arginine, R) of the prion 
protein gene (PRNP) is associated with an increased 
risk of disease development (HUNTER et al., 1989, 
1997a,b; BELT et al., 1995; DAWSON et al., 1998). 
It is well established that the ARR/ARR genotype’s 
susceptibility to the agents causing scrapie is 
extremely low, although no longer considered null 
(GROSCHUP et al., 2007). On the other hand, the 

VRQ allele is considered to be associated with high 
susceptibility (HUNTER et al., 1997a; ELSEN et 
al., 1999; ACÍN et al., 2004). The PRNP genotype 
also affects the pathogenesis of scrapie and the 
tissue levels and distribution of PrPSc in affected 
sheep. For instance, in ARR/VRQ sheep, there is 
little or no involvement of the lymphoid tissue in 
agent replication (ANDRÉOLETTI et al., 2000; 
VAN KEULEN et al., 2008). 

Even though the first record of scrapie in 
Brazil occurred in a Hampshire Down ovine 
imported from England (FERNANDES et al., 
1978), and other cases were reported sporadically 
(RIBEIRO, 1996; DRIEMEIER, 1998) or notified at 
World Organization for Animal Health - OIE 
(http://www.oie.int), the clinical cases in 2003 and 
2004 (POHL DE SOUZA et al., 2005) from the 
flock reported here were the first cases considered 
autochthonous in Brazil. After this, other scrapie 
cases have been reported and published 
(http://www.oie.int; RIBEIRO et al., 2007; 
ANDRADE et al., 2011). Some of these cases were 
on the animals from Paraná State (SOTOMAIOR et 

Received: 11/04/14 
Accepted: 15/10/14 



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Biosci. J., Uberlândia, v. 31, n. 4, p. 1189-1199, July/Aug. 2015 

al., 2012). 
The ovine population of Paraná totals about 

639,000 animals. Although it represents about 4.5% 
of the Brazilian sheep population, the Paraná sheep 
population increased 27.5% from 2006 to 2012 
(IBGE, 2012). Several foreign breeds are kept in 
Paraná such as Suffolk, Hampshire Down, Ile de 
France, Texel, and in recent years, Dorper. Other 
indigenous Brazilian breeds such as Santa Inês 
(McMANUS et al., 2010) are also bred and mixed 
with the former mentioned breeds. In these breeds, 
seven different genotypes have been reported in 
sheep from Paraná (SOTOMAIOR et al., 2008), the 
most frequently found one being ARQ/ARQ (41%), 
followed by ARR/ARQ (29%). 

Although plenty of data demonstrate the 
association between genotype and resistance or 
susceptibility to scrapie, differences among breeds 
and herds are evident, indicating that the use of 
uniform criteria for selection cannot necessarily be 
applied for different sheep breeds, flocks, or even 
countries (LÜHKEN et al., 2004; ACÍN et al., 
2004). In Brazil, although there is not yet a breeding 
program to control scrapie, some data on PRNP 
polymorphisms have been published (LIMA et al., 
2007; PACHECO et al., 2007; SOTOMAIOR et al., 
2008; PASSOS et al., 2008; ANDRADE et al., 2011; 
IANELLA et al., 2012; SANTOS et al., 2012). 
These articles mainly give the percentages of the 
different polymorphisms, but do not make any kind 
of association with the presence of the PrPSc.  

The purpose of this study was to 
retrospectively analyze the PRNP polymorphisms 
found in an ovine flock in which three animals had 
been diagnosed in 2003 and 2004 with the classical 
form of scrapie, and to determine if these 
polymorphisms are associated with the presence of 
PrPSc. Since the whole flock was slaughtered in 
2006, we were able to examine lymphoid and 
central nervous tissues by immunohistochemistry 
(IHC) for altered prion protein (PrPSc). 

 
MATERIAL AND METHODS 
 
Flock 

All 128 animals studied belonged to the 
sheep-breeding herd of the Pontifical Catholic 
University of Paraná (PUCPR) Experimental Farm 
located in the city of Fazenda Rio Grande, State of 
Paraná, Brazil. This flock consisted of two groups of 
animals: 53 pure Hampshire Down (HD) animals 
and 75 crossbred (CR) animals, from different 
breeds such as Texel, Ile de France, Hampshire 
Down, and even animals with no recognizable 
breed. Additionally, there were 5 crossbred animals 

of the Creolle (2), Polwarth (1), Corriedale (1), and 
Karakul (1) breeds. Pure HD rams were used in 
crossings with the crossbred females. In the 
purebred flock, except for four older ewes (born 
between 1997 and 1999) and the rams, all other 
animals had been born at PUCPR. As for the 75 
crossbred animals, 33 (44%) of them had been 
bought from other farms, during 2002 and 2003, and 
the remaining animals had been born at PUCPR. 

 
Flock history 

Until January 2003, no animal had 
presented signs compatible with scrapie. After this 
time point, between 2003 and 2004, three animals 
(females, aged between 6 and 6 ½ years, Hampshire 
Down) with clinical signs were isolated in the 
veterinary hospital of PUCPR, the official public 
veterinary service was informed, and sick animals 
were euthanized after observation (Pohl de Souza et 
al., 2005; 
http://www.agricultura.gov.br/arq_editor/file/Aniam
al/programa%20nacional%20dos%20herbivoros/SC
RAPIE.pdf). These three animals were officially 
diagnosed with classical scrapie. After almost 3 
years, genotyping and immunohistochemistry were 
performed on the whole flock in 2006, after the 
ending of a legal dispute. 
 
DNA extraction and purification 
 DNA was extracted from frozen whole 
blood samples. Four mL of blood was collected 
from the external jugular using Vaccutainer® tubes 
with ethylenediamine tetraacetic acid (EDTA) as an 
anticoagulant. DNA extraction and purification was 
performed using proteinase K and the 
phenol/chloroform method. 
 
PCR-RFLP 
 For the analysis of restriction fragment 
length polymorphisms (RFLP), polymerase chain 
reaction (PCR) was performed on the DNA samples 
according to the methods described in Lühken et al. 
(2004). For amplification of the DNA fragments, 
reactions were performed in a total volume of 30 
µ L, using 1X reaction buffer, 1.5 mM magnesium 
chloride, 0.2 mM dNTP, 10 pmol of each primer, 
0.6 U Taq DNA Polymerase, and 2.0 µ L of sample 
DNA (variable concentration). PCR amplification 
conditions were as follows: an initial denaturation 
temperature of 94°C for 60 s, followed by 40 cycles 
at 94°C for 20 s, 55°C for 20 s, 72°C for 20 s, and a 
final extension at 72°C for 5 min. In both PCR 
reactions, the forward primers were the same (5´-
TGTGGCAGGAGCTGCTGCAGCT–3´). The 
reverse primers were 5´-



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Biosci. J., Uberlândia, v. 31, n. 4, p. 1189-1199, July/Aug. 2015 

TGCACAAAGTTGTTCTGGTTACTATC–3´ 
(RP1), and 5´-
GCACAAAGTTGTTCTGGTTACTATAT-3´ 
(RP2), for the first and second reactions, 
respectively. The amplified regions correspond to 
nucleotides 342 to 539 (first reaction) and 
nucleotides 342 to 538 (second reaction) of the 
PRNP, with lengths of 197 and 196 base pairs (bp) 
respectively. 
 Amplified products from each reaction were 
digested with restriction enzymes BspDI and BspHI. 
The first PCR product (197 bp) was cleaved with 
BspHI, while the second (196 bp) was doubly 
digested with BspDI and BspHI. Fifteen µ L 
reactions (1X buffer, 2.5 U enzyme, 8 µ L PCR 
product, and ultra-pure water) were kept in an oven 
at 37°C overnight (12h on average). Subsequently, 
the total volume of the cleavage reaction was 
separated by electrophoresis in a 2.5% agarose gel 
prepared with TBE buffer. The electrophoretic run 
was carried out at 35 V for 6 h. Following staining 
with ethidium bromide (0.5 µ g/mL), the gel image 
was processed using Gel-Pro Analyzer 4.0 software 
(Media Cybernetics, Rockville, MD) for genotype 
analysis. 
 Amplification with primer RP1 creates an 
artificial restriction site for BspHI, when the codon 
for histidine is at position 171. Similarly, 
amplification with RP2 creates an artificial 
restriction site for BspDI, when the codon for 
arginine is at position 171. In both the fragments, 
the codon for valine at position 136 and that for 
histidine at position 154 are restriction sites for 
BspHI. Therefore, digestion of the 197 bp PCR 
product with BspHI and the double digestion of the 
196 bp product with BspHI and BspDI enzymes 
generate two band patterns that together define the 
genotypes of the PRNP for analysis of their alleles. 
 
Diagnosis of PrP

Sc
 by immunohistochemistry 

 Immunohistochemistry (IHC) was 
performed in 111 sheep (17 had died of different 
causes). Those 111 animals were slaughtered in 
2006, and their organs were collected for subsequent 
analysis to determine the presence of PrPSc through 
IHC. The collected organs included the brain, the 
third eyelid, the tonsils, the ileum, and the spleen. 
 The fragments were kept in 10% 
formaldehyde and analyzed by 
immunohistochemistry using monoclonal antibody 
F89/160.1.5 (O´Rourke et al., 1998) according to 
the protocol used by the Federal University of Rio 
Grande do Sul. Samples were treated with 

proteinase K (DAKO, Ready-to-use) diluted in 
distilled water (1:1) to eliminate the PrPC isoform. 
Positive and negative controls were available. 
 
Statistical analysis 
 Statistical analysis of the relationships 
between the different genotypes and the presence or 
absence of PrPSc was performed by building 
contingency tables and using the chi-square (χ2) 
method, with the Yates correction and the Fisher’s 
exact test. Additionally, we used the Z test to 
compare proportions between the different 
genotypes in the Hampshire Down animals and in 
the crossbred animals. 
 
RESULTS 
 
Genotyping 

Six genotypes were found in the 128 
genotyped animals: ARR/ARR, ARR/ARQ, 
ARQ/ARQ, ARR/VRQ, ARQ/VRQ, and 
ARQ/AHQ (Table 1). The genotype ARR/ARQ was 
the most frequent (45.3%) followed by ARQ/ARQ 
(28.1%), ARR/ARR (14.1%), and ARQ/VRQ 
(8.6%). ARR/VRQ and ARQ/AHQ had a genotype 
frequency of less than 2.5%. When comparing 
crossbred animals (CR) with Hampshire Down 
(HD) animals, CR showed a greater percentage of 
ARR/ARR genotypes than HD (21.3% vs. 3.8%, 
respectively), the same with ARQ/VRQ (2.7% vs. 
1.9%) and ARQ/AHQ (2.7% vs. 0%). The HD breed 
had greater proportion of the genotypes ARR/ARQ 
(49.1% vs. 42.7%), ARQ/ARQ (32.1% vs. 25.3%) 
and ARQ/VRQ (13.2% vs. 5.3). Results from 25 
samples were validated by DNA sequencing (data 
not shown), and in all cases, the results confirmed 
the genotype suggested by RFLP analysis. 
PrP

Sc
 diagnosis by immunohistochemistry 

 
The results of the IHC analyses of the 111 

animals of the infected flock showed that the 95 
samples (85.6%) were negative for the presence of 
PrPSc in all the examined organs and tissues. Sixteen 
animals (14.4%) were positive for one or more 
tissues, 8 of which were pure Hampshire Down 
(17% of 47 pure animals examined) and 8 were 
crossbred (12.5% of 64 crossbred animals 
examined). Figure 1 shows the labeling by IHC in 
the tonsil of one of the positive animals. It is 
important to observe that at the moment that they 
were killed, no animals presented any clinical signs. 

 
 



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Biosci. J., Uberlândia, v. 31, n. 4, p. 1189-1199, July/Aug. 2015 

Table 1. Number of animals (n), according to genotype and breed of the scrapie-infected flock 
 

Breed 
 

ARR/ARR 
 

ARR/ARQ 
Genotype 

ARQ/ARQ 
 

ARR/VRQ 
 

ARQ/VRQ 
 

ARQ/AHQ 
Hampshire Down 2 26 17 1 7 0 
Crossbred 16 32 19 2 4 2 
Total 18 58 36 3 11 2 

 
 

 
Figure 1. Immunohistochemical assay of a tonsil lymphoid follicle showing positive PrPSc immunolabeling , 

Scale bar = 50 µm. 
 
The 16 positive sheep showed differences in 

their reactive organs and tissues (Table 2). No 
animal was positive for all the tissues examined, and 
tonsil was the tissue with the highest percentage of 
reactive samples: 81.25%. Only one animal was 
positive in the brain (6.25%). The average age was 
4.3 years, ranging from 3 years up to 7 years. Only 
one (6.25%) of the positive samples was from a 
male, but males represented less than 15% of the 
total animals examined. From the 16 positive 
animals, 5 (31.25%) were not born in the PUCPR´s 
farm (Table 2). 
 
Relationship between genotyping and IHC 

When the 16 positive samples by IHC are 
compared to the animals’ genotype, it is possible to 

observe that animals from three genotypes were 
positive for PrPSc. The highest percentage of 
positive animals had the ARR/ARQ genotype, 
representing 56.25% of the positive samples (n = 9), 
followed by the ARQ/ARQ genotype with 31.25% 
(n = 5). Genotype ARQ/VRQ represented 12.5% of 
the PrPSc positive animals (n = 2). In genotypes 
ARR/ARR, ARR/VRQ, and ARQ/AHQ, there were 
no positive cases (Table 3). 

When we compare the proportion of each 
genotype in the total number of positive and 
negative samples (Table 3), there was a significant 
difference only for genotype ARR/ARR. The 
frequencies of genotypes ARR/VRQ and 
ARQ/AHQ were insufficient for comparison. 

 
 



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Biosci. J., Uberlândia, v. 31, n. 4, p. 1189-1199, July/Aug. 2015 

Table 2. Immunohistochemistry (IHC) results from 16 positive animals for the presence of PrPSc, according to 
breed (Hampshire Down (HD) and crossbred (CR)), genotype, positive tissues, year of birth, and 
location of birth 

Breed Genotype Positive tissues in IHC Year of birth Location of birth 

HD ARQ/ARQ Spleen and brain 1999 Other 
HD ARQ/ARQ Spleen and tonsil 2002 PUCPR 

HD ARQ/ARQ Spleen and third eyelid 2003 PUCPR 

HD ARR/ARQ Tonsil 2003 PUCPR 
HD ARR/ARQ Spleen and ileum 2003 PUCPR 
HD ARR/ARQ Tonsil 2003 PUCPR 
HD ARQ/VRQ Tonsil 2001 PUCPR 
HD ARQ/VRQ Tonsil 2001 PUCPR 
CR ARQ/ARQ Tonsil 1999 Other 
CR ARQ/ARQ Tonsil 2002 PUCPR 
CR ARR/ARQ Tonsil 1999 Other 
CR ARR/ARQ Spleen and tonsil 2001 PUCPR 
CR ARR/ARQ Tonsil 2001 Other 
CR ARR/ARQ Tonsil 2001 Other 
CR ARR/ARQ Tonsil 2002 PUCPR 
CR ARR/ARQ Tonsil and ileum 2003 PUCPR 

 HD = Hampshire Down CR = crossbred 
 
In the chi-square (χ2) method, we could not 

find an influence by genotype in the positive and 
negative animals, when considering all data together 
(HD + CR), or only HD breed data. For the 
crossbred animals, there was a statistically 
significant influence of genotype in the IHC result. 

The ARR/ARQ genotype was significantly different 
(p < 0.05) from the other genotypes. The ARR/ARQ 
genotype corresponds to 39.3% of the negative and 
to 75% of the positive results by IHC (Table 3). The 
proportional differences between the remaining 
genotypes were not statistically significant.  

 
Table 3. Comparison of animals with positive (+) and negative (-) immunohistochemistry (IHC) for the 

presence of PrPSc by genotype of Hampshire Down (HD) and crossbred (CR) ovine in the PUCPR 
flock  

Genotype HD 
+ IHC  
n (%) 

CR 
+ IHC  
n (%) 

Total 
+ IHC  
n (%) 

HD 
- IHC  
n (%) 

CR 
- IHC  
n (%) 

Total 
- IHC  
n (%) 

ARR/ARR 0 (00.0) 0 (00.0) 0 (00.0) 2 (05.1) 16 (28.6) 18 (18.9) 
ARR/ARQ 3 (37.5) 6 (75.0) 9 (56.2) 19 (48.7) 22 (39.3) 41 (43.2) 
ARR/VRQ 0 (00.0) 0 (00.0) 0 (00.0) 1 (02.6) 2 (03.6) 3 (03.2) 
ARQ/ARQ 3 (37.5) 2 (25.0) 5 (31.3) 12 (30.8) 11 (19.6) 23 (24.2) 
ARQ/VRQ 2 (25.0) 0 (00.0) 2 (12.5) 5 (12.8) 3 (05.3) 8 (08.4) 
ARQ/AHQ 0 (00.0) 0 (00.0) 0 (00.0) 0 (00.0) 2 (03.6) 2 (02.1) 

Total 
number of 

animals 
8 (100.0) 8 (100.0) 16 (100.0) 39 (100.0) 56 (100.0) 95 (100.0) 

 
DISCUSSION 

 
The clinical cases of scrapie of the flock 

reported here were the first notification of scrapie in 
native, not imported animals, in a Brazilian flock of 
sheep 

(http://www.agricultura.gov.br/arq_editor/file/Ania
mal/programa%20nacional%20dos%20herbivoros/S
CRAPIE.pdf) and the further genotyping and 
immunohistochemistry of the whole flock allowed 
us to conduct a retrospective study relating genotype 
and resistance to scrapie in an ovine flock in Brazil. 



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Biosci. J., Uberlândia, v. 31, n. 4, p. 1189-1199, July/Aug. 2015 

These cases also led the Brazilian Ministry of 
Agriculture to reconsider categorizing scrapie as an 
exotic disease in the country. The disease is now 
part of the National Encephalopathy Program. 
Nowadays, in cases of suspect alive animals, 
samples from third eyelid or other lymphoid tissue 
are examined. All positive IHC for PrPSc are 
considered a scrapie-positive diagnosis (BRASIL, 
2008). 

In the flock studied here, up to the moment 
the animals were killed, none had presented any 
clinical signs. However, out of the 111 animals 
slaughtered and tested, 16 were positive for scrapie 
(14.4% of the total). The concern about the presence 
of such asymptomatic PrPSc carrier sheep is that 
they may transmit the disease agent for long 
periods, before clinical cases appear in the flock. 
Billinis et al. (2004), also testing healthy animals in 
a herd where there were positive cases of scrapie, 
found 25% of animals in the latent phase (without 
clinical signs) compared to the clinically healthy. 
Tongue et al. (2005), in a survey of the prevalence 
of PrPSc in 14 herds whose animals were slaughtered 
after the confirmation of cases of scrapie, estimated 
a prevalence of 6.6%, ranging from zero to 15.4%, 
in clinically healthy appearing animals. 

In cases of classical scrapie, infection occurs 
by the oral route via infection of the Peyer’s patches 
followed by replication in the gut-associated 
lymphoid tissues (GALT). It may then spread to the 
central nervous system through the autonomic 
nervous fibers innervating the digestive tract 
(ANDRÉOLETTI et al., 2000). Environmental 
contamination can also occur through the placenta 
and infected fetal fluids (ANDRÉOLETTI et al., 
2002; DETWILER; BAYLIS, 2003; TOUZEAU et 
al., 2006). It is known that during the scrapie 
incubation period, ewes with the susceptible 
genotypes accumulate large quantities of PrPSc and 
may transmit the agent through the placenta even in 
the first gestation, well before the beginning of the 
clinical signs of the illness (ANDRÉOLETTI et al., 
2002). There is also the possibility of milk 
transmission, as described by Konold et al. (2008). 
The environmental contamination in the case of this 
flock reported here could have been caused by the 
three ewes, earlier diagnosed with scrapie, since 
they gave birth many times before the appearance of 
any clinical signs. Thus, the infection of all the 
animals born on the farm probably occurred soon 
after their birth, due to the contaminated 
environment. However, there were also positive 
animals from the group that was bought from other 
farms, and were contaminated only as adults. 
Working with the introduction of adult, scrapie-free 

ovine in contaminated flocks, Ryder et al. (2004) 
showed the possibility of horizontal transmission in 
adult animals. Their work also shows that, even if 
young animals are more susceptible to scrapie than 
adult animals, sheep are susceptible to infection at 
different ages, including adulthood. Such data 
corroborate the results found in the present work, in 
which animals born in other farms and introduced in 
the contaminated flock were positive by IHC. In the 
crossbred flock, 33 (44%) were over 1-year old 
when they were brought into the flock. Four of them 
were positive by IHC, indicating that these animals 
(12.12%) were probably infected when they were 
adults. When adult animals are infected, the slower 
PrPSc dissemination would be due to the important 
role of the ileal Peyer's patch. In experimental 
infections, Heggebo et al. (2003) showed that 5 
weeks after the challenge, lambs with the 
susceptible genotypes already presented coloration 
in the Peyer's patch, indicating the important role of 
that lymphoid tissue in the absorption and 
dissemination of the scrapie agent. If infection 
occurs after the involution of this organ, it may be 
less effective and lead to slower development of the 
illness (ERSDAL et al., 2003). 

Of the tissues and organs examined from the 
16 confirmed cases, the tonsil presented the highest 
positive reaction percentage (81.25%). Schreuder et 
al. (1998) and Andréoletti et al. (2000) report PrPSc 
accumulation in biopsies of tonsils, independent of 
the animal’s age. It is interesting to observe that one 
animal presented a positive result from the obex, 
without any clinical signs. In the clinical phase of 
the illness, Andréoletti et al. (2000) found PrPSc not 
only in the central nervous system (CNS) but also in 
the lymphoid tissues of VRQ/VRQ animals. 
However, in ARR/VRQ animals, the PrPSc 
deposition occurred only in the CNS. Other authors 
have reported similar data in natural infections in 
Texel ovine (Van KEULEN et al., 1996; 
SCHREUDER et al., 1998), which led them to 
suggest that ovine carrying an ARR allele would not 
accumulate PrPSc in the lymphoid tissues 
(ANDRÉOLETTI et al., 2000; Van KEULEN et al., 
2008). These data are not in accordance with the 
results found in the present work, which showed 
that 56.25% of the positive results were from 
animals heterozygous for the ARR allele, and that 
all had positive signals in the tonsil or spleen. Ersdal 
et al. (2003) also found a scrapie positive 
ARR/VRQ lamb in the Peyer’s patch on day 86 of 
life, the same age found for another VRQ/VRQ 
lamb, indicating that even in the presence of the 
ARR allele, there can be prion accumulation in the 
lymphoid tissue. 



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Biosci. J., Uberlândia, v. 31, n. 4, p. 1189-1199, July/Aug. 2015 

In the analysis of the genotypes associated 
with PrPSc accumulation, the presence of PrPSc in 
ARR/ARR, which represented 14.1% of animals, 
was not found. Although there are confirmed cases 
of ARR/ARR animals with classical scrapie 
(Groschup et al., 2007), the susceptibility of the 
ARR/ARR genotype to classical scrapie is 
considered very low (Hunter et al., 1997a; Elsen et 
al., 1999; Acín et al., 2004). 

Analyzing the data on pure-HD animals, the 
genotypes with positive cases were ARQ/ARQ 
(37.5% of the cases), ARR/ARQ (37.5%), and 
ARQ/VRQ (25%). In the HD breed, as in the 
Suffolk breed, alanine is the amino acid most 
frequently found at codon 136 (Dawson et al., 
1998), and the presence of the VRQ allele is absent 
or rare. In sheep from Paraná, Sotomaior et al. 
(2008) found 2% of ARR/VRQ and 10% of 
ARQ/VRQ genotypes in the HD breed. In other 
Brazilian HD breed samples, Passos et al. (2008) 
found a frequency of 6% for allele VRQ and Ianella 
et al. (2012) also found the ARR/VRQ genotype 
(4.2%). Andrade et al. (2011), working with 
Suffolk, found the VRQ allele at a frequency of 3%. 
Therefore, in these breeds, the ARQ allele is 
considered to be the most susceptible to scrapie 
(HUNTER et al., 1997a; DAWSON et al., 1998), as 
seen in this study, where all the positive animals 
were at least heterozygous for the ARQ allele. 

In the analysis of the crossbred animals, 
although there was no significant difference, there 
were more positive than negative cases within the 
ARQ/ARQ genotype, as noted in the HD breed. 
However, when the ARQ allele is associated with 
the ARR allele, this genotype represents 75.0% of 
the cases positive for the presence of PrPSc and 
39.3% of the cases of negative animals (p < 0.05). 
These results differ from that usually presented in 
the literature (O´DOHERTY et al., 2002; BAYLIS 
et al., 2004) about the influence of alleles, which 
must be studied more deeply. The fact that the 
animals in the present study come from the 
crossbreeding of various breeds may have 

contributed to these results, since there are 
differences in genotype susceptibility in different 
breeds. Authors working with the Texel breed found 
the ARQ allele to be codominant with the VRQ 
allele, and therefore did not grant protection (Belt et 
al., 1995), but this is contrary to data from the Ile de 
France breed (LAPLANCHE et al., 1993). 

In modern production, selection of animals 
that are genetically resistant to diseases is an 
ongoing effort. Thus, determination of the genetic 
basis of a disease is needed, as it allows for the 
definition of more objective criteria for efficient 
breeding and crossbreeding systems. Since many 
differences among breeds and herds are seen in their 
resistance or susceptibility to scrapie, the use of 
uniform criteria for selection cannot necessarily be 
applied to different sheep breeds, flocks, or 
countries (LÜHKEN et al., 2004; ACÍN et al., 
2004). Thus, defining the relationship between 
genotype and resistance or susceptibility is essential 
before starting any type of controlled breeding 
program. Even though this is a retrospective study 
from a flock analyzed in 2006, the present work 
may contribute to future studies and scrapie control 
programs, since it associates different genotypes 
with the presence of PrPSc in crossbred animals and 
in the Hampshire Down breed, although the number 
of positive animals was quite low. 

It is also necessary that before a selective 
breeding program is proposed, the previous 
structure of the population and the degree of 
variability of the PRNP among different breeds is 
known. Currently, data about PRNP polymorphisms 
in Brazil are being published (LIMA et al., 2007; 
PACHECO et al., 2007; SOTOMAIOR et al., 2008; 
PASSOS et al., 2008; ANDRADE et al., 2011; 
IANELLA et al., 2012; SANTOS et al., 2012). 
Further studies like these must be carried out in 
order to identify the resistant and susceptible 
genotypes in the different breeds, contributing to 
increase the value of rams and dams, which may be 
commercialized with the certification of scrapie 
resistance and will help control the disease. 

 
 

RESUMO: Scrapie nos ovinos está associada a pelo menos três polimorfismos do gene da proteína priônica 
celular (PRNP) nos códons 136, 154 e 171. Países onde o scrapie é endêmico têm utilizado programas de melhoramento, 
com a seleção para os alelos mais resistentes. Há alguns dados disponíveis de genotipagem do PRNP em ovinos no Brasil, 
e o scrapie tem sido observado esporadicamente desde 1978. O Paraná é o Estado brasileiro onde a maioria dos casos de 
scrapie foi diagnosticada. Um rebanho, que teve três casos clínicos de scrapie em 2003 e 2004, foi genotipado (128 ovinos 
- 53 Hampshire Down e 75 mestiços) e abatido (111 ovinos - 47 Hampshire Down e 64 mestiços) em 2006. Amostras de 
tecido linfóide e sistema nervoso central foram examinadas por imunohistoquímica (IHQ) para presença de proteína 
priônica alterada (PrPSc). Seis genótipos foram encontrados nos 128 animais genotipados: ARR/ARQ foi o mais frequente 
(45,3%), seguido por ARQ/ARQ (28,1%), ARR/ARR (14,1%) e ARQ/VRQ (8,6%). ARR/VRQ e ARQ/AHQ 
apresentaram menos de 2,5% de freqüência do genótipo. Na IHC, 16 animais com exame positivo para a presença da 
proteína priônica celular alterada (PrPSc) foram detectados. As tonsilas foram o tecido com a mais alta porcentagem de 



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amostras reativas: 81,25% do total das amostras positivas. Considerando os 16 animais positivos, nove (56,25%) tinham o 
genótipo ARR/ARQ, seguido pelo genótipo ARQ/ARQ com 31,25% (n = 5) e ARQ/VRQ com 12,5% (n = 2). Todos os 
animais positivos estavam clinicamente saudáveis, representando, portanto, 14,14% de casos pré-clínicos de scrapie neste 
rebanho. 

 
PALAVRAS - CHAVE: Ovis aries. Prion. Imunohistoquímica. Genotipagem. 

 

 
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