Trop Anim Health Prod DOI 10.1007/s11250-015-0849-9

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Report of outbreaks of classical scrapie in Dorper sheep and associated prion protein gene polymorphisms in affected flocks Caroline Pinto de Andrade 1 & Eduardo Conceição de Oliveira 1 & Juliano Souza Leal 1 & Laura Lopes de Almeida 2 & Luiza Amaral de Castro 1 & Sergio Ceroni da Silva 1 & David Driemeier 1

Received: 11 December 2014 / Accepted: 11 May 2015 # Springer Science+Business Media Dordrecht 2015

Abstract Scrapie is an infectious neurodegenerative disease affecting sheep and goats, related with conformational alteration of an isoform of the prion protein that leads to deposition and aggregation in the host’s central nervous system. Occurrence of the natural disease can be influenced by host genetic factors, such as a single nucleotide polymorphism of the prion protein gene. This study reports three scrapieaffected Dorper flocks located on three different farms in Brazil. The objective of this study was to analyze these three flocks using scrapie diagnostics, combining histology, immunohistochemistry, genotyping, and western blot assays. For immunohistochemistry, 192 sheep were selected and 308 sheep blood samples were taken for genotyping. A total of 22 sheep were scrapie positive by immunohistochemistry. Of these, four presented clinical signs and had scrapie immunoreactivity at the obex in western blot assays. The sheep without clinical signs were positive in lymphoid organs, such as the third eyelid and rectal mucosa. The major genotypes found on the flocks were ARQ/ARQ, ARQ/ARR, and ARQ/ VRQ for codons 136, 154, and 171. Most of the sheep were considered to be at moderate to high risk, based on risk groups for developing scrapie. Some blood samples were sequenced, and polymorphisms were identified in other codons, such as 127, 142, and 143. Our data demonstrate the importance of

* Caroline Pinto de Andrade [email protected] 1

Setor de Patologia Veterinária, Departamento de Patologia Clínica Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Porto Alegre, RS 91540-000, Brazil

2

Instituto de Pesquisas Veterinárias Desidério Finamor (IPVDF), Estrada Municipal do Conde, 6000 Eldorado do Sul, RS, Brazil

preclinical scrapie diagnosis in Brazilian sheep, as most of the affected sheep showed no clinical signs, and emphasize the relevance of genotyping other Dorper sheep to determine the genotypic profile of the breed. Keywords Outbreak . Dorper . Scrapie . Prion . Immunohistochemistry . Real-time PCR

Introduction Scrapie is a transmissible spongiform encephalopathy of sheep and goats that causes progressive neurological deterioration and death (Woolhouse et al. 1998). It is characterized by central nervous system (CNS) accumulation of an abnormal isoform (PrPSc) of a host-encoded cellular prion protein (PrPc) (Hunter et al. 2008). The major polymorphisms of the prion protein gene (PRNP) associated with susceptibility or resistance to the infection are at codons 136 (A or V), 154 (R or H), and 171 (R, Q, or H) (Clouscard et al. 1995; Hunter et al. 1996). The PRNP genotypes VRQ/VRQ, ARQ/VRQ, and ARQ/ARQ are associated with high scrapie susceptibility, while the ARR/ARR genotype exhibits resistance to infections with classical scrapie (Baylis et al. 2004; Hunter et al. 1996). Polymorphisms at other codons can also be involved in infection resistance or susceptibility, such as polymorphism in scrapie-affected ovines at codon 143 (Acín et al. 2004). In addition, a polymorphism at codon 142 has been described in sheep and is associated with a longer disease incubation period (Vaccari et al. 2007). Other codon alterations have been identified, including codons 141 (atypical scrapie), 112, 127, 137, and 176 (Acín et al. 2004; Benestad et al. 2008;

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Goldmann et al. 1996; Laegreid et al. 2008; Vaccari et al. 2006, 2009). The first case of classical scrapie in Brazil was diagnosed in 1978 (Fernandes et al. 1978). Since then, 16 cases of scrapie have been reported from 1978 to 2010 (Sotomaior et al. 2012). Our previous work described a classical scrapie in Suffolk sheep where one animal presented clinical signs of infection (Andrade et al. 2011). Until now, no cases of scrapie have been reported in Dorper and White Dorper sheep. Additionally, although some work has characterized PRNP polymorphisms in Brazilian flocks, few studies report these polymorphisms in association with scrapie outbreaks (Andrade et al. 2013; Ianella et al. 2012; Lima et al. 2007; Pacheco et al. 2007; Passos et al. 2008; Santos et al. 2012; Sotomaior et al. 2008). The present study describes three outbreaks of classical scrapie occurring in Dorper and White Dorper flocks on three different rural properties in Brazil. To characterize these outbreaks, we analyzed PRNP polymorphisms at codons 136, 154, and 171 and performed immunohistochemical analysis of the obex and lymphoid tissues of exposed and affected animals.

Materials and methods Epidemiological background for the outbreaks On the first property (hereafter referred to as Boutbreak 1^) located in southern Brazil, one sheep was diagnosed with the classical form of scrapie based on immunohistochemical analysis, clinical signs, and histopathological findings (index case). An epidemiologic survey of this property was performed by the Brazilian Ministry of Agriculture (MAPA) and Parana State Department of Agriculture (SEAB) to identify animals that may have been exposed directly or indirectly to the affected sheep. This property produced purebred Dorper and White Dorper sheep; it was also home to female Santa Inês, Suffolk, and Texels, which were used as recipients. The sheep selected for testing had potential contact with the affected sheep and included animals born in the same stall or to the same receptor animal as the affected animal. Data collected for each animal included sex, age, registry number, and degree of purity. On the second property (called Boutbreak 2^), one Dorper sheep was diagnosed with clinical signs of scrapie (diagnosis confirmed by histopathology and immunohistochemistry). This occurred approximately 1 year after outbreak 1 at a property in southern Brazil. An epidemiologic survey found that there had been animals from outbreak 1 on the second property, which were purchased after outbreak 1 had occurred.

Two years after outbreak 1, a third outbreak (called Boutbreak 3^) was identified, located in the southern of Brazil. Three animals at this property had been obtained from the site of outbreak 1 (after outbreak occurred), and one of these showed clinical signs. In three properties, the sheep selected for diagnosis were older than 1 year of age, as indicated in national regulations, where, according to rules, the sample collection occurs in exposed animals with over 12 months of age. Animals, sample collection, and DNA preparation In outbreak 1, the samples from the third eyelid and rectal mucosa were taken from 75 sheep (71 females and 4 males) from a flock of 335 Dorper/White Dorper sheep for antemortem scrapie diagnosis. Blood samples were also collected from 181 animals for real-time polymerase chain reaction (PCR) genotyping, including the 75 animals. In outbreak 2, the samples from the third eyelid, rectal mucosa, and blood were collected from all 74 animals in the flock, which consisted of crossbred Dorper sheep. In outbreak 3, the blood samples were collected from the entire flock (53 Dorper sheep) and also 43 samples from the third eyelid and rectal mucosa. Rectal biopsies were collected after application of 1 % lidocaine hydrochloride in the rectum, using an insulin syringe (Leal et al. 2012). Third eyelid collection was performed using 1 % tetracaine hydrochloride and 0.1 % phenylephrine drops as previously described (O’Rourke et al. 2002). For histological examination, 3-μM sections were stained with hematoxylin and eosin (HE). Animals with scrapie immunoreactivity in the biopsy samples were euthanized, and the obex was collected for later histological, immunohistochemical, and western blot analyses. Clinical signs were identified by questioning owners or attending veterinarians at the time of sample collection. Peripheral blood samples were collected for PCR, sequencing, and genotyping using EDTA vacutainer tubes. Genomic DNA was extracted from 500 μL of total blood, using a commercially available kit and following the manufacturer’s instructions. Immunohistochemistry After paraffin embedding, tissue sections were dried for 12 h at 60 °C, deparaffinized, and treated with 10 % hydrogen peroxide in methanol for 20 min. Sections were then incubated with formic acid for 5 min and washed in TBS, followed by proteinase K treatment for 40 s. PrPSc immunohistochemistry was performed using the primary anti-PrP monoclonal antibody mAb F99/97.6.1 and anti-PrP monoclonal antibody mAb P4. The antibodies were diluted 1:500 in physiologic buffered saline (PBS) prior to use, and the sections were

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incubated for 18 h at 4 °C, followed with a biotinylated secondary antibody for 20 min and with peroxidase-conjugated streptavidin for 20 min. Tissues were exposed to chromogen diaminobenzidine (DAB) for 1 min or 3-amino-9ethylcarbazole (AEC) counterstained with hematoxylin for 30 s. Each experiment included control sections of obex and lymphoid tissues from scrapie-affected and healthy control sheep. For confirmation of one positive ovine (ARR/ARR), PrPSc immunohistochemistry was performed in another laboratory at the Institute of Novel and Emerging Diseases (Friedrich Loeffler Institute) with the anti-PrP monoclonal antibody mAb 2G11. Western blot The cooled obex tissue samples were used for PK digestion from affected sheep with clinical signs, which were confirmed by histopathological and immunohistochemical (IHC) analyses in accordance with established procedures (Vaccari et al. 2006). The samples were submitted to 12 % SDS-PAGEj under reducing conditions and transferred to nitrocellulose membranes for western blot analyses. The primary monoclonal antibody utilized was mAb P4 at a 1:500 dilution in PBS, biotinylated secondary antibody, and peroxidase-conjugated streptavidin. The staining was performed with DAB. Negative control healthy ovine, bovine, and affected sheep without clinical sign of tissue infection were produced in the same assays. Primers, PCR, and sequencing conditions Real-time PCR for PRNP codon 136, 154, and 171 genotyping was performed as described previously from the first working group (Andrade et al. 2013). Sheep with less common genotypes, animals with polymorphisms that had not been previously identified in our laboratory, and animals that were positive by immunohistochemistry were sequenced for genotype confirmation as described below. In total, 43 samples were sequenced from outbreak 1, 21 samples from outbreak 2, and 18 samples from outbreak 3. PCR reactions were performed using a forward primer flanking codon 136 and a reverse primer flanking codon 171, generating a 245-bp amplicon (L’Homme et al. 2008). PCR reactions contained 15 pmol of each primer, 1.5 mM MgCl2, 200 μM dNTPs, 1× Platinum Taq buffer, 1 U Platinum Taq DNA polymerase, and 1 μL of genomic DNA. PCR reactions were performed using the following parameters: 95 °C for 5 min followed by 35 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s and a final step at 72 °C for 10 min. PCR products were purified using the PCR purification kit and quantified using a fluorescence quantification system according to the manufacturer’s instructions. PCR products were sequenced using the BigDye Terminator in an ABI PRISM

3130. The Phred quality score of at least 20 was obtained for each individual base in the consensus sequence (Ewing and Green 1998; Ewing et al. 1998). The resulting chromatograms were analyzed using the Staden package version 1.7.0 programs and novoSNP (Staden et al. 2003; Weckx et al. 2005). Genotyping analysis The real-time PCR data from the sheep samples were used to derive genotype frequencies using the formula fij =nij /N, where fij corresponds to the ij genotype frequency, nij corresponds to the number of animals with the genotype ij, and N corresponds to the total number of animals analyzed. These results were used to classify sheep according to the risk groups described by Dawson et al. (2008). Furthermore, the possible association between the variables, genotype, and positive immunohistochemistry were analyzed using the chi-square test (χ2) (p1 >1 >1 >1 >1

Third eyelid Third eyelid Rectal mucosa Obex Rectal mucosa Rectal mucosa Third eyelid/rectal mucosa Rectal mucosa Rectal mucosa Third eyelid/rectal mucosa Obex Obex Third eyelid/rectal mucosa Third eyelid, rectal mucosa, obex Rectal mucosa Third eyelid/rectal mucosa Rectal mucosa

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

− − − + − − − − − − + + − + − − −

3 5 3 3 3 1 3 3 3 2 3 3 5 3 2 2 3

3.6 3.7 3.8 3.9 3.10

Female Female Female Female Female

>1 >1 >1 >1 >1

Third eyelid/rectal mucosa Rectal mucosa Third eyelid/rectal mucosa Rectal mucosa Rectal mucosa

ARQ/ARQ ARQ/ARR ARQ/ARR ARQ/ARQ ARQ/ARR

− − − − −

3 2 2 3 2

a

Born at the same time as the animal with clinical signs

b

Lambed in the same bay where the animal with clinical signs born

c

Obtained from the property in outbreak 1

Fig. 1 Histopathology of the obex of a sheep with scrapie with vacuolization adjacent to neurons. a, b Case 1.4; c, d case 2.8 (see Table 1). Magnification: a, c ×200; b, d ×400. Staining with hematoxylin and eosin

Trop Anim Health Prod Fig. 2 Immunohistochemistry of the obex of a sheep with scrapie with antibody mAb F99/97.6.1. a, b Case 2.7 (see Table 1). PrPSc was detected in the cytoplasm of neurons using AEC as a chromogen. c, d Case 3.2 (see Table 1). PrPSc was detected in the cytoplasm of neurons using DAB as a chromogen. Magnification: a, b, d ×400; c ×200

Genotypic analysis determined that of the four immunohistochemistry-positive sheep, two had genotype ARQ/ARQ, one had genotype VRQ/VRQ, and one had genotype ARQ/ARH. All of these genotypes are associated with moderate to high susceptibility

(Fig. 4). Of the three scrapie-negative sheep that were considered high risk based on exposure to the infected animal, two had genotype ARQ/ARQ. Only one sheep had genotype ARQ/ARR, which is considered low risk of infection (Fig. 4).

Fig. 3 Immunohistochemistry of lymphoid follicles from the rectal mucosa and third eyelid with antibody mAb F99/97.6.1 and antibody mAb P4. PrPSc was detected using DAB as a chromogen a Case 1.2, b case 1.3, c case 2.2, d case 2.6, e case 3.1, and f case 3.3 with antibody

mAb F99/97.6.1. g Case 1.2, h case 1.3, i case 2.2, j 2.6, k 3.1, and l 3.3 with antibody mAb P4 (see Table 1). PrPSc was detected using DAB as a chromogen. Magnification: a, b, e–j, l ×200; c, d, k ×400

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Outbreak 1 (Dorper/White Dorper)

50.0%

Outbreak 2 (Dorper crossbred) Outbreak 3 (Dorper)

Genotypes Frequencies

40.0%

30.0%

20.0%

10.0%

0.0% ARR/ARR

ARR/AHQ

1 (very low)

ARR/ARH

ARR/ARQ

AHQ/ARQ

2 (low)

ARH/ARH

ARH/ARQ

3 (moderate)

ARQ/ARQ

ARR/VRQ 4 (moderate)

ARQ/VRQ

VRQ/VRQ

5 (high)

Risk groups (genotypes)

Fig. 4 Distribution of animals within risk groups based on genotypes for codon 136/154/171 in sheep from three outbreaks (n=181, 74, and 53 for outbreaks 1–3, respectively). The risk groups correspond to 1 (very low), 2 (low), 3 (moderate), 4 (high moderate), and 5 (high)

The frequency of genotypes within the flock was also determined, and the most prevalent was ARQ/ARQ with 46.4 %, followed by ARQ/ARR with 25 % and ARQ/VRQ with 15 %. The most resistant genotype ARR/ARR was present at 3.8 %. Based on this analysis, both the Dorper/White Dorper and crossbred sheep appear to be susceptible to the disease due to the high frequency of susceptible genotypes in the flock (Fig. 4). When we classified sheep into risk groups, 17.7 % of purebreds and 15.5 % of crossbred sheep were classified as risk group 5 (highly susceptible). Fifty percent of purebreds and 53 % of crossbred sheep were classified into risk groups 3 and 4 (moderately susceptible), and 30 % of purebreds and 22.2 % of crossbred sheep were risk groups 1 and 2 (low susceptibility) (Fig. 4). Samples from positive animals and animals with less frequent genotypes were sequenced for confirmation to identify SNPs at other codons. At codon 142, the allelic frequencies were 73.3 % (II), 23.3 % (IT), and 3.4 % (TT). One immunohistochemistry-positive animal had changes in these codons, resulting in the substitution of an isoleucine for a threonine. A polymorphism at codon 143 was observed in crossbred animals; the heterozygous form of the allele (HR) was present at a frequency of 10 %. Furthermore, no polymorphisms were identified at codon 141. Analysis of outbreak 2 Of the 74 samples from outbreak 2, eight were positive by immunohistochemistry and were sacrificed, generating a frequency of 10.8 % positives in the flock (Table 1). All of the

sheep tested were approximately 2-year-old Dorper females. Two positive animals showed clinical signs (disturbances in movement) and vacuolization of neurons in the obex consistent with scrapie (Fig. 1c, d, see Table 1). The histopathological lesions in these cases were similar to those in outbreak 1 with PrPSc immunolabeling in the neurons of the obex (Fig. 2a, b, see Table 1). The other sheep had no clinical signs or histological lesions but presented positive staining in third eyelid and rectal mucosa (Fig. 3c, d, i, j, see Table 1). The genotype ARQ/ARQ (risk group 5) was found in six of the positive animals, including both animals with clinical signs. The remaining two positive animals had genotypes ARQ/ARR and ARR/ARR. The PrPSc immunohistochemistry of the ARR/ARR-positive animal also was confirmed in another laboratory, where the results were identical to our PrPSc immunohistochemistry. The most frequent genotype in the flock was ARQ/ARQ with 53 %, followed by ARQ/ARR with 24.3 % and ARQ/ VRQ with 5.4 %. The ARR/ARR genotype was present in 4 % of the flock. Classification of these sheep into risk groups indicates that the flock is considered susceptible; 66.2 % of the animals are classified as moderate to high risk, and 33.8 % is considered low risk for infection (Fig. 4). Similar to outbreak 1, sequencing was performed on the samples and identified a rare polymorphism at codon 127 that resulted in a substitution of a serine for a glycine with an allelic frequency of 14 % in ARQ/ARQ sheep (including one positive sheep). No polymorphism was identified at codon 141.

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Analysis of outbreak 3 Of the 43 samples from the third property, 10 sheep were positive by immunohistochemistry and were sacrificed, generating a frequency of 23.3 % positive in the flock (Table 1). Only one showed clinical signs (difficulty in locomotion), and this animal was originally from the property involved in outbreak 1. This sheep had histological and immunohistological findings with PrPSc immunolabeling in the neurons of the obex (Fig. 2c, d, see Table 1). In animals without clinical signs, the PrPSc immunolabeling in the third eyelid and/or rectal mucosa was performed (Fig. 3e, f, k, l, see Table 1). Genotype analysis found four positive animals with the ARQ/ARQ genotype (risk group 3) and one with the ARQ/ VRQ genotype (risk group 5); all are considered susceptible. The other positive sheep had the ARQ/ARR genotype, which is associated with low susceptibility (Fig. 4). In the flock, the genotype analyses showed that the ARQ/ARR genotype is the most frequent (41.5 %), followed by 39.6 % with ARQ/ARQ and 11.3 % with ARR/ARR. Risk group analysis divided this flock into low-risk (54.7 %) and moderate-risk (45.3 %) groups (Fig. 4). No polymorphisms were found in other codons by sequencing, including codon 141.

Western blot The western blot with monoclonal antibody mAbP4 allows discrimination of classic scrapie, BSE, and the atypical. The positive obex tissue samples of affected sheep with clinical signs produced a molecular pattern of PrPSc characterized by four bands migrating between 25 and 48 kDa, compatible with the classical form of scrapie (Fig. 5). The major 12-kDa band, which was observed in the atypical scrapie samples, was not

Fig. 5 Western blot with antibody mAb P4 of proteinase K digestion PrPSc from the obex of sheep and bovines. Lane 1 molecular weight marker (MWM), lanes 2–5 scrapie-affected sheep with clinical signs, lane 6 sheep with preclinical scrapie (lymphoid organ positive), lane 7 healthy ovine, lane 8 healthy bovine

found in our test. Furthermore, the negative controls did not present any molecular banding patterns. Statistical analyses When analyzing the association of genotypes with positive immunohistochemistry for scrapie through chi-square analysis, we found no association of genotypes related to positive immunohistochemistry. For the association between the frequencies of positive results for scrapie by immunohistochemistry among the flocks, we observed significant differences between herds 1 and 3 (p=0.003). However, the comparison of flocks 1 and 2 and flocks 2 and 3 showed no significant differences between frequencies. No difference was found when analyzing the association of risk groups with positive sheep.

Discussion This study describes three outbreaks of scrapie in Dorper/ White Dorper sheep. Our data are consistent with those of other publications reported by Heaton et al. (2010) and Ianella et al. (2012), which show that breeds are genetically susceptible to scrapie infection. In the three outbreaks, we found ARQ/ARR sheep with positive lymphoid tissues. In addition, to validate the study, we performed immunohistochemistry with two specific antibodies to confirm the results. In both analyses, it was possible to detect PrP S c immunolabeling in the lymphoid tissues of positive animals. One study reports the occurrence of three ARQ/ARR-positive sheep to scrapie, 1086 days after the infection by detecting prion in the lymphoreticular tissue. Gonzalez et al. (2014) believe that the susceptibility of ARQ/ARR sheep is linked to a combination of factors, such as the dose of infection and the age. Another study reported by Toungue et al. (2006) showed that during the periods 1998–2002, in the national scrapie plan of Great Britain, ARQ/ARR ovines with a frequency of 0.34 % were found, indicating that the resistance of ARQ/ARR scrapie sheep is not absolute. Several factors must be considered, for example, specifying the susceptibility of each race, the degree of virulence of the strain, and environmental stress. Antibody P4 was utilized to confirm that sheep were infected with the classical form of scrapie based on the fact that the banding patterns of PrPSc between classical and atypical form of scrapie, in western blot, are different. The bands between 25 and 35 kDa were found in the fresh obex-positive samples, and upper bands >40 kDa were found in FFPE obex tissue and represent prion protein dimers, observed by Loicano et al. (2010) and Soto et al. (2005). The affected scrapie sheep without clinical signs did not produce band patterns, most likely because the prion was not present in the

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central nervous system, which has been demonstrated by O’Rourke et al. (2002). In addition, both healthy ovines and bovines were negative by western blot, indicating that normal prion protein is totally degraded after proteinase K treatment. In accordance with Thuring et al. (2004), the western blot analyses allowed differentiation of classical and atypical scrapie and BSE. Comparing positive immunohistochemistry with genotyping to assess whether the genotype was related to the positivity by chi-square analysis, we found no significant difference into account, in the tested genotype combinations. These results suggest that we cannot associate the positivity with scrapie genotype alone but must take into account the strain, the degree of exposure to the agent, and livestock management. Analyzing the three codons from the three outbreaks, the ARQ/ARQ genotype was most common in Dorper sheep at 46 % (mean of three flocks). This result is similar to the data found in national publications reported by Ianella et al. (2012) and Sotomaior et al. (2008), where the percentage of Dorper sheep was between 50 and 63 %. The second most frequently genotype was ARQ/ARR at 30 % (mean of three flocks), a result that was also described by Ianella et al. (2012). The VRQ/ARQ genotype was found in 8 % (mean of three flocks), which differs from other studies described by Ianella et al. (2012) and Sotomaior et al. (2008) in which 20–22 % had this genotype. The VRQ/ARR genotype was found in 4.9 %; a similar result was obtained by Sotomaior et al. (2008), which observed this genotype in 4 % of a flock. The remaining genotypes were found in 1 to 5 % in the flocks and were not found in other works, most likely because the number of sheep analyzed in other studies was much lower compared to ours. Comparing the three outbreaks studied, the risk groups were found to be at low and moderate risk, which is consistent with the results found by Sotomaior et al. (2008), showing that 70 % of a herd of Dorper sheep belonged to the same risk groups. In the three outbreaks described, we observed a large number of susceptible sheep regardless of breed. At codon 171, considering the most relevant to susceptibility, approximately 90 % of sheep examined had genotype QR or QQ. This finding is consistent with data obtained by Ianella et al. (2012) and Sotomaior et al. (2012) who found that between 90 and 100 % of the Dorper sheep in Brazil presented these genotypes. When the sheep were classified into risk groups, approximately 69 % of animals in Dorper/White Dorper flocks from these three outbreaks belonged to moderate- to high-risk groups. This result is consistent with study results reported by Ianella et al. (2012) and Sotomaior et al. (2008), which showed that Dorper flocks are 70 % moderate to high risk. However, the latter study identified 11 % as low susceptible risk, but in our work and in Ianella et al. (2012), 30 % of Dorper/White Dorper sheep were low susceptible risk. Only one of the sheep positive for scrapie had the genotype ARR/ARR, which is associated with infection resistance.

Therefore, it was tested with two antibodies and PrPSc immunolabeling was positive. To confirm and validate the ARR/ARR-positive ovine, the materials were sent to another laboratory and the results were positive for scrapie. Few studies reported by Andréoletti et al. (2002), Buschmann et al. (2004), Groschup et al. (2007), and Jeffrey et al. (2014) described the occurrence of classical scrapie in sheep with this genotype, suggesting that there is no absolute genetic resistance. Some rare polymorphisms in codons 127, 142, and 143 were found through sequencing. The largest number of polymorphisms in codons 142, 143, 154, and 171 was found in crossbred animals, suggesting that crossbreeding may contribute to an increased rate of polymorphisms. One positive animal presented a polymorphism at codon 127. This polymorphism has been previously described by Goldmann (2008) and Vaccari et al. (2009) in Mongolian and Chinese sheep, but the effect on scrapie susceptibility has not yet been defined. One genetically highly susceptible (VRQ/VRQ) sheep that was positive by immunohistochemistry presented a polymorphism at codon 142. The effect of this alteration is not yet known but has been described in sheep in New Zealand by Wang et al. (2008). One polymorphism also was found at codon 143 and had been previously identified in other flocks observed by Acín et al. (2004), DeSilva et al. (2003), Loicano et al. (2010). The study described by Vaccari et al. (2007) suggests that the heterozygous form (H143R) might offer protection against infection in goats. All data suggest that Dorper sheep are more susceptible to infection, and even sheep that have low genetic susceptibility may acquire scrapie. These animals most likely suffered a high pressure for prion-contaminated environment, which may have led to an increase in the number of infected animals. The fact is that studies in Dorper sheep should continue, making a larger survey of the genotypic frequency of breedassociated infection and verifying if there is a more resistant genotype that can assist in future breeding programs.

Acknowledgments The authors would like to thank all of their collaborators at the Ministério da Agricultura do Estado do Paraná and the Secretaria da Agricultura do Paraná. This work was supported by a fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Proc. 578226/2008-1, and CNPq, Proc. 505917/ 2008-4). The first author (C.P. de Andrade) was supported through a fellowship from the postdoctoral process number of CNPq 501863/ 2013-3. The second author (Eduardo C. de Oliveira) was also supported by Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS/CAPES) with fellowship process number 830-25.51/13-7. We would also like to thank Prof. Dr. Martin H. Groschup of the Institute of Novel and Emerging Diseases (Friedrich Loeffler Institute) for collaborating on PrPSc immunohistochemistry. Conflict of interest The authors declare that they have no competing interests.

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Report of outbreaks of classical scrapie in Dorper sheep and associated prion protein gene polymorphisms in affected flocks.

Scrapie is an infectious neurodegenerative disease affecting sheep and goats, related with conformational alteration of an isoform of the prion protei...
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