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Romanian Biotechnological Letters Vol. 16, No. 4, 2011 Copyright © 2011 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER

6419

The scrapie genetic susceptibility of some sheep breeds in southeast Romanian area and genotype profiles of sheep scrapie infected

Received for publication, January 31, 2011

Accepted, August 4, 2011

M. R. OTELEA(1), M. ZAULET(2), DUDU A.(2), F. OTELEA(4), S. BARAITAREANU(1), D. DANES(1,3) (1)University of Agronomical Science and Veterinary Medicine Bucharest, Faculty of Veterinary Medicine, 105 Splaiul Independentei, 5th district Bucharest, Romania, E-mail: [email protected], [email protected], [email protected] (2)University of Bucharest, Molecular Biology Center, 91-95 Splaiul Independentei, 5th district Bucharest, Romania, E-mail: zaulet_mihaela @yahoo.com, tel. 0040213181575, fax 0040213181575/ 102 (3)Institute of Comparative Medicine, University of Agronomical Science and Veterinary Medicine Bucharest, Splaiul Independentei 105, 050097, Bucharest, Romania (4) Braila County Veterinary Laboratory, Romania, 344 Calea Galati, tel.0239.610.689, fax 0239.610.691, email:[email protected]

Abstract The paper describes the PRNP genotype in a traditional Romanian sheep farm, officially confirmed with scrapie by National Reference Laboratory for Transmissible Spongiform Encephalopathies (NRL-TSE, IDAH Bucharest, Romania), in 2007. It also presents the usually methods of scrapie screening in Romania counties and the PRNP gene polymorphisms of PrPsc positive sheep, by DNA sequencing. The results proved the high variation of susceptibility in non-genetically selected herd. Even more, the number of rams susceptible and highly susceptible to scrapie was significant (29%), making difficult the genetic selection. All immunoenzymatic and histopathological positive results were confirmed by IHC and immunoblotting performed by NRL-TSE. These results support the reliability of screening methods used in active surveillance of scrapie in Romania. The sequencing analysis confirmed the diversity of sheep PRNP genetics. It was detected one new single nucleotide polymorphism at codon 185 (Iso-Thr substitution) and two frequently reported substitutions at codons 143 (His-Arg substitution) and 171 (His-Gln substitution). The results of this study support the use of sequence-based genotype investigations in developing the sheep-breeding programs. Keywords: genotyping, TSEs, polymerase chain reaction (PCR), sequencing Introduction Scrapie is the transmissible spongiform encephalopathy (TSE) in small ruminants, including sheep (Ovis aries), domestic goat (Capra aegagrus hircus) and European mouflon (Ovis ammon musimon), produced by some protein infectious particles named prions [13, 16, 20, 29, 30]. The posttranslational modification of the normal cellular host-derived PrPC into an abnormal protease-resistant PrPSc isoform (prion) represents the main feature of these fatal neurodegenerative diseases [15, 25, 27, 29]. TSEs refer to a group of fatal neurodegenerative disorders affecting mammals and human beings [23, 29, 32]. TSEs affecting human beings include Creutzfeldt-Jakob-disease, Gerstmann-Sträussler-Scheinker-syndrome (GSS), Kuru, fatal familial insomnia (FFI) and sporadic fatal insomnia (SFI). TSEs affecting mammals are scrapie in sheep and goats, transmissible mink encephalopathy (TME), chronic wasting disease in elk, mule deer and white-tailed deer (CWD), bovine spongiform encephalopathy in cattle (BSE, mad cow disease), exotic ungulate encephalopathy in exotic ungulates (EUE) and feline spongiform encephalopathy in cats (FSE) [32].

M. R. OTELEA, M. ZAULET, DUDU A., F. OTELEA, S. BARAITAREANU, D. DANES

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The first European scrapie reports were in England (1732) and Germany (1759). Romanian official report of scrapie was in 2002 [43]. Disease often occurs in animals aged 3-4 years and rarely under 18 months age. Field and experimental studies suggest that the spread of scrapie among sheep occurs mainly by horizontal transmission. Rams role in disease spread is recognized often the disease occurrence being related with the purchase of breeding rams [28]. The salivary glands were investigated for the presence of the pathological prion protein (PrPSc) in scrapie-affected sheep and healthy controls, and PrPSc was detected both, in naturally and experimentally infected sheep [44]. Scrapie was the first spongiform encephalopathy whose transmissibility was proved [10]. Genetics studies revealed a strong association between classical scrapie and PRNP polymorphisms at codons 136, 154 and 171 [7, 17, 18, 22, 33, 39, 42, 43, 48, 49]. The combination of this polymorphism gives rise to 5 PRNP codon haplotypes or alleles and 15 PRNP diploid genotype combinations, commonly found in sheep. The sheep susceptibility to scrapie varies widely between genotypes: from greatest resistance (ARR/ARR) to extreme susceptibility (VRQ/VRQ) [3, 4, 17, 18]. According to the UK National Scrapie Plan, the sheep genotype susceptibility is classified in 5 classes [12]. The sheep-genotype scrapie resistant is ARR/ARR (Class 1). The sheep genetic ARR/AHQ, ARR/ARH, ARR/ARQ (Class 2) are resistant to scrapie but they need a special attention in selection programs. The sheep with lower genetic resistance to scrapie are ARQ/ARH, ARQ/AHQ, AHQ/AHQ, ARH/ARH, AHQ/ARH, ARQ/ARQ (Class 3). The sheep sensitive to scrapie are ARR/VRQ (Class 4), and the highest sensitive are AHQ/VRQ, ARH/VRQ, ARQ/VRQ, VRQ/VRQ (Class 5). The main purpose of the study was to present the PRNP genotype in a traditional Romanian sheep farm, official confirmed with scrapie by National Reference Laboratory for Transmissible Spongiform Encephalopathies (IDAH Bucharest, Romania), in 2007. The study has two secondary objectives, to present the usually methods of scrapie screening in Romania counties and to describe the PRNP gene polymorphisms of PrPsc positive sheep by DNA sequencing. Materials and Methods Biologic material Blood samples, randomly collected from 845 sheep have been submitted to the study of ovine prion protein gene (PRNP) genotypes. The samples originated from scrapie contaminated farm, some of them even from subjects exhibiting clinical signs of disease. All blood samples were collected in 3 ml tubes containing K3-EDTA and stored at -20°C before DNA extraction. Brainstems samples collected from 11 sheep PrPSC-positive were genotyped and the ovine prion protein gene (PRNP) was sequenced. All positive-samples were priory tested by ELISA and histological examined in Braila County Veterinary Laboratory. The official diagnostic of scrapie was performed in National Reference Laboratory for Transmissible Spongiform Encephalopathies (NRL-TSE, IDAH Bucharest, Romania) by immunohistochemistry and immunoblotting, according with Regulation (EC) 999/2001 [42], and the outbreak was reported in 2007 to World Animal Health Information Database (WAHID) [45]. Histological examination used only sections of medulla oblongata at the level of obex, in accordance with sampling recommendation of OIE Terrestrial Manual 2009 [46]. The rapid immunodiagnostic test used fresh medulla taken at the obex or just caudal to the obex, in accordance with the manufacturer’s instructions.


Romanian Biotechnological Letters, Vol. 16, No. 4, 2011 6421

DNA extraction Samples were processed using the High Pure PCR Template Preparation Kit (Roche, Mannheim, Germany) for isolation of nucleic acids from mammalian whole blood (200 µl) and mammalian tissue (200 µl brain homogenate) for LightCycler®PCR. The kit was used in accordance with the manufacturer’s instructions. Genotyping of ovine prion protein gene (PRNP) variants by PCR with melting curve analysis The protocol used in this study was as described by LightCycler® Scrapie Susceptibility Mutation Detection Kit (TIB MOLBIOL, Berlin, Germany), used with Roche FastStart reagents LightCycler® FastStart DNA MasterPLUSHybProbe (Roche, Mannheim, Germany). Melting curve analysis was performed using LightCycler® 2.0 Real-Time PCR System (Roche, Germany). Prion Protein Gene Sequencing Amplification of extracted DNA. The PCR was set up in a volume of 20µl with 5µl DNA solution, volume of reagents/reaction: 0.1µl Ampli GoTaq Polymerase- Promega (5U/µl), 1.5µl MgCl2 (25mM), 2µl dNTP mix (10mM), 2.5 µl buffer 10x for polymerase, 0.6µM of forward primer (5'-GGTCAAGGTGGTAGCCACAGTCAGTGGAAC-3') and 0.6µM of reverse primer (5'-ATCACCCAGTACCAGAGAGAATCCCAGGCT-3'). The thermal cycling program included: a denaturation (10 min at 95°C), 40 cycles of amplification (30s at 95°C, 30s at 59°C, 60s at 72°C), and a final extension (10 min at 72°C). It was used an iCycler Thermal Cycler from Bio-Rad. Agarose gel electrophoresis. PCR products (5µl) were separated on a 2% agarose gel (80V for 45min) containing ethidium bromide in TBE buffer (10mM Tris, 2.75g boric acid/l, 1mM Na2 EDTA). The visualization was performed in UV transilluminator and the images were captured with a Polaroid camera and video documentation system (Bioprofil-Vilber Lourmat). The length of amplified fragment is 402 pb. Sequencing. The PCR reaction products were purified with Wizard® PCR Preps DNA Purification System (Promega, Madison, WI, USA), and then the concentration and purity of the products were evaluated by spectrophotometry (Eppendorf BioPhotometer, Hamburg, Germany). The DNA sequencing reactions were done using BigDye Terminator Kit v3.1 (Applied Biosystems). The precipitation of DNA sequencing product was performed with BigDye XTerminator® Purification Kit, (Applied Biosystems, Foster City, CA, USA). The primers used for sequencing were the same as for the PCR amplification. The sequencing was performed on 3130 Genetic Analyzer (Applied Biosystems). The obtained sequences were manually edited and aligned using CLUSTAL W application from Bio Edit software with a reference sequence from GenBank database (accession number U67922). In vitro purification and detection of PrPSc by immunoenzymatic (sandwich format) technique The immunoenzymatic technique (EIA) used the TeSeE Purification and Detection Kit (Bio-Rad, Marnes-la-Coquette, France). The protocol of vitro purification and detection of PrPSc was performed according with the manufacturer’s instructions. Histopathological examination The medulla oblongata fragments were formalin-fixed, embedded in paraffin, cut at 4µm and haematoxylin-eosin stained. The histological exam was performed in accordance with the method used at the European TSE Community Reference Laboratory, VLA Weybridge [47]. Note: Our study used the results of NRL-TSE (IDAH Bucharest, Romania) only to confirm the results of active surveillances. NRL-TSE performs immunohistochemistry and



immunoblotting in accordance with the method used at the European TSE Community Reference Laboratory, VLA Weybridge [47]. Results and Discussions The sheep PRNP gene genotyping by PCR with melting curve analysis revealed the presence of all five classes of scrapie susceptibility (fig. 1). Most of sheep were in class 2 (48%), meaning sheep genetic resistant to scrapie, but asking a special attention to be used in selection programs. In class 1 (genotype ARR/ARR) we found 142 sheep (17%), from this only 11 rams. This group could be used to create a new herd, but precautions should be taken to prevent transmission of scrapie by secondary sources, looking to the experimentally transmission study results [34]. In the same herd were identified 218 (26%) sheep in class 3, 28 (3%) sheep in class 4 and 45 (6%) sheep in class 5.

Figure 1. The scrapie genetic risk classes distribution. In the corner: the frequency in rams In previously Romanian genotyping studies, focused on specific sheep breed (Turcana breed, Sibian ecotype), were obtained similar results in the herd structure about males: 19, 25% genotype ARR/ARR class 1, but different results in other classes: 27.68% class 2; 40.69%% class 3; 5.53% class 4 and 6.84% class 5 [9]. In studies performed in herds from outside countries the data are heterogeneous for all classes: the genotype ARR/ARR (class 1) frequency has 21.3% in Great Britain, 15, 9% in Norway, 11% in Canada and 9.3% in Japan [4, 17, 19, 22]. Vaccari G. et al. confirmed the correlation between scrapie and (Gln/Gln) 171 in breeds with no variation for Ala136 [42]. All haplotypes have been found in the studied herd, but not in all breeds. The ARQ variant was the most frequent in all breeds, haplotype frequencies ranging between 50.0% and 100 % in half breed of Merinos (HMB) and half breed of Milk Line of Merinos (HMLMB), respectively. The VRQ variant has not been found in HMLMB and Turcana Breed (TB), but 20% of HMB and 18.9% of Lacaune breed (LB) show this haplotype. The ARR variant was present in the studied herd, frequencies ranging from 0.0% to 23.1 %, as in HMLMB and Suffolk Breed (SB), respectively. The VRQ haplotype frequency in the herd was 8.9%, this being highly similar with data previously published, where this haplotype missed or registered low rate [1, 2, 3, 8, 18, 19, 35, 38].



Table 1. Frequency of scrapie susceptibility classes* and PRNP genotypes** in healthy sheep of some Romanian breeds

MB MLMB HBM MMB HMLMB LB SB TBHB TB Breed*** No. % No. % No. % No. % No. % No. % No. % No. % No. %

Total

Total 128 100 137 100 20 100 70 100 6 100 159 100 13 100 299 100 2 100 834 Classes

1 23 18.0 21 15.3 4 20.0 12 17.1 0 0.0 24 15.1 3 23.1 52 17.4 2 100.0 141 2 65 50.8 67 48.9 7 35.0 31 44.3 3 50.0 62 39.0 5 38.5 161 53.8 0 0.0 401 3 30 23.4 36 26.3 5 25.0 24 34.3 3 50.0 43 27.0 4 30.8 73 24.4 0 0.0 218 4 4 3.1 10 7.3 1 5.0 1 1.4 0 0.0 10 6.3 0 0.0 3 1.0 0 0.0 29 5 6 4.7 3 2.2 3 15.0 2 2.9 0 0.0 20 12.6 1 7.7 10 3.3 0 0.0 45

Haplotypes ARR 23 18.0 21 15.3 4 20.0 12 17.1 0 0.0 24 15.1 3 23.1 52 17.4 2 100.0 141 AHQ 8 6.3 5 3.6 0 0.0 3 4.3 0 0.0 8 5.0 0 0.0 2 0.7 0 0.0 23 ARH 1 0.8 6 4.4 2 10.0 1 1.4 0 0.0 1 0.6 0 0.0 6 2.0 0 0.0 17 ARQ 86 67.2 92 67.2 10 50.0 51 72.9 6 100.0 96 60.4 9 69.2 226 75.6 0 0.0 579 VRQ 10 7.8 13 9.5 4 20.0 3 4.3 0 0.0 30 18.9 1 7.7 13 4.3 0 0.0 74

Genotypes ARR/ARR 23 18.0 21 15.3 4 20.0 12 17.1 0 0.0 24 15.1 3 23.1 52 17.4 2 100.0 141 ARR/AHQ 5 3.9 5 3.6 0 0.0 3 4.3 0 0.0 8 5.0 0 0.0 2 0.7 0 0.0 23 AHQ/AHQ 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 ARR/ARH 1 0.8 6 4.4 2 10.0 1 1.4 0 0.0 1 0.6 0 0.0 5 1.7 0 0.0 16 AHQ/ARH 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 ARR/ARQ 59 46.1 56 40.9 5 25.0 27 38.6 3 50.0 53 33.3 5 38.5 154 51.5 0 0.0 362 AHQ/ARQ 3 2.3 3 2.2 0 0.0 3 4.3 0 0.0 9 5.7 1 7.7 1 0.3 0 0.0 20 ARH/ARH 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 1 0.3 0 0.0 1 ARH/ARQ 1 0.8 1 0.7 0 0.0 1 1.4 0 0.0 2 1.3 0 0.0 5 1.7 0 0.0 10 ARQ/ARQ 26 20.3 32 23.4 5 25.0 20 28.6 3 50.0 32 20.1 3 23.1 66 22.1 0 0.0 187 ARR/VRQ 4 3.1 10 7.3 1 5.0 1 1.4 0 0.0 10 6.3 0 0.0 3 1.0 0 0.0 29 AHQ/VRQ 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 ARH/VRQ 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 2 0.7 0 0.0 2 ARQ/VRQ 6 4.7 3 2.2 3 15.0 2 2.9 0 0.0 20 12.6 1 7.7 7 2.3 0 0.0 42 VRQ/VRQ 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 1 0.3 0 0.0 1

Total 128 100 137 100 20 100 70 100 6 100 159 100 13 100 299 100 2 100 834 *In accord with UK National Scrapie Plan classification. **Genotypes were obtained by PCR with melting curve analysis. *** Breeds: MB, Merinos breed; MLMB, Milk Line of Merinos Breed; HBM, Half breed of Merinos; MMB, Meat Line of Merinos Breed, HMLMB Half breed of Milk Line of Merinos Breed; LB, Lacaune Breed; SB, Suffolk Breed; TBHB, Tigae with Black Head Breed, TB, Turcana Breed. Table 2. Frequency of scrapie susceptibility classes and PRNP genotypes in scrapie-positive sheep of some Romanian breeds

MB MLMB HBM MMB HMLMB LB SB TBHB TB Breed No. % No. % No. % No. % No. % No. % No. % No. % No. %

Total

Total 3 100 0 0 2 100 2 100 1 100 0 0 0 0 3 100 0 0 11 Classes

1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 3 100 0 0 2 100 2 100 0 0 0 0 0 0 3 100 0 0 10 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0 0 0 0 0 0 0 1 100 0 0 0 0 0 0 0 0 1

Haplotypes ARR 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 AHQ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ARH 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ARQ 3 100 0 0 2 100 2 100 0 0 0 0 0 0 3 100 0 0 10 VRQ 0 0 0 0 0 0 0 0 1 100 0 0 0 0 0 0 0 0 1

Genotypes ARR/ARR 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ARR/AHQ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 AHQ/AHQ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ARR/ARH 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 AHQ/ARH 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ARR/ARQ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 AHQ/ARQ 0 0 0 0 0 0 1 50 0 0 0 0 0 0 0 0 0 0 1 ARH/ARH 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ARH/ARQ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ARQ/ARQ 3 100 0 0 2 100 1 50 0 0 0 0 0 0 3 100 0 0 9 ARR/VRQ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 AHQ/VRQ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ARH/VRQ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ARQ/VRQ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 VRQ/VRQ 0 0 0 0 0 0 0 0 1 100 0 0 0 0 0 0 0 0 1

Total 3 100 0 0 2 100 2 100 1 100 0 0 0 0 3 100 0 0 11



In the herd it has been identified 12 genotypes of PRNP gene. The genotypes AHQ/AHQ, AHQ/ARH, AHQ/VRQ have not been identified, but the genotype AHQ/AHQ was reported in previously Romanian study [9]. The frequencies of each genotype in nine breeds from the scrapie infected herd are shown in table 1 (healthy sheep) and table 2 (scrapie-positive sheep). In the studied herd, the frequency of PRNP risk genotypes, susceptible and highly

susceptible to scrapie (classified in risk groups 4 and 5, respectively), have been at 3.4% ARR/VRQ, 0.2% ARH/VRQ, 5% ARQ/VRQ, 0.2% VRQ/VRQ, 0.1% of them corresponding to subjects scrapie-positive (table 2). PRNP genotypes also associated with scrapie susceptibility (risk group 3) showed a frequency of 2.5% AHQ/ARQ, 0.1% ARH/ARH, 1.1% ARH/ARQ, and 23.2% ARQ/ARQ, with 1.1% of them being those subjects scrapie-positive (table 2).

(a) MB

(b) MLMB

(c) HBM

(d) MMB

(e) HMLMB

(f) LB

(g) SB

(h) TBHB

(i) TB

Figure 2. PRNP genotypes of some Romanian sheep breeds

Table 3. Frequency of scrapie susceptibility classes and PRNP genotypes in healthy and scrapie-positive of some Romanian sheep breeds

Healthy Scrapie No. % No. %

Scrapie (%)/ Healthy(%)

Classes 1 141 16.7 0 0.0 0.00 2 401 47.5 0 0.0 0.00 3 218 25.8 10 1.2 0.05 4 29 3.4 0 0.0 0.00 5 45 5.3 1 0.1 0.02

Total no. of animals = 845 Genotypes

ARR/ARR 141 16.7 0 0.0 0.00 ARR/AHQ 23 2.7 0 0.0 0.00 AHQ/AHQ 0 0.0 0 0.0 0.00

ARR/ARH 16 1.9 0 0.0 0.00

AHQ/ARH 0 0.0 0 0.0 0.00 ARR/ARQ 362 42.8 0 0.0 0.00 AHQ/ARQ 20 2.4 1 0.1 0.05 ARH/ARH 1 0.1 0 0.0 0.00 ARH/ARQ 10 1.2 0 0.0 0.00 ARQ/ARQ 187 22.1 9 1.1 0.05 ARR/VRQ 29 3.4 0 0.0 0.00 AHQ/VRQ 0 0.0 0 0.0 0.00 ARH/VRQ 2 0.2 0 0.0 0.00 ARQ/VRQ 42 5.0 0 0.0 0.00 VRQ/VRQ 1 0.1 1 0.1 1.00

Total no. of animals = 845



The scrapie-positive sheep from this investigated herd, harboured the PRNP genotypes ARQ/ARQ (n=9) or AHQ/ARQ (n=1), both belonging to risk group 3, and only one scrapie-positive animals with haplotype VRQ (VRQ/VRQ genotype) were found. These data are in line with those reported in Italian sheep breeds [2, 24], but differ from other reports whose analysis frequently detected VRQ/VRQ and VRQ/ARQ genotypes in scrapie-infected sheep [11, 36]. That could be a similar status as those described by the Italian study[2], where it was found a peculiar genetic target of the scrapie strains circulating, or is the result of ARQ/ARQ genotype high frequency in the herd (table 3). The explication of this situation needs further investigations. Referring to the usually methods of scrapie screening in Romania counties, all herd animals were tested by the immunoenzymatic technique (EIA). The exam revealed 11 scrapie-positive sheep and one goat [45]. In our study, all positive sheep were investigated by histopathological exam, genotyping and prion protein gene sequencing. The PRNP gene polymorphisms of PrPsc positive sheep by DNA in the scrapie-affected sheep group, have been shown two haplotypes (ARQ, VRQ) and three genotypes (AHQ/ARG, ARQ/ARQ, and VRQ/VRQ) with three associated mutations (Fig. 6, 7, and 8). By 11 positive animals, 10 sheep were in class 3 with genotype ARQ/ARQ (3 MB, 2 HBM, 1 MMB, and 3 TBHB) and AHQ/ARQ (1MMB), and one in class 5 with genotype VRQ/VRQ (1 MMB). Those results are in concordance with the data previously published, in which the frequency of genotype ARQ/ARQ is very high [1, 2]. The histopathological exams matched with the EIA results, and NRL-TSE confirmed scrapie diagnosis by immunohistochemistry and immunoblotting. NRL-TSE is using the same methods for scrapie diagnostic as the European TSE Community Reference Laboratory, VLA Weybridge [47]. Those results make confident the Romanian scrapie survey program.

Figure 3. Histopathological aspects in Merinos sheep with clinical signs scrapie-associated. The spongiform aspect of the neuropil and degenerescence, atrophy and lyses in neuronal perykaria (medulla oblongata at the level of obex, x400, HE). In the corner: neuronal perykaria retracted, with pyknosis and one vacuole (x1000, HE)

Figure 4. Histopathological aspects in Merinos sheep with clinical signs scrapie-associated. The spongiform aspect of the neuropil and lyses in neuronal perykaria. HE (medulla oblongata at the level of obex, x630, HE). In the corner: normal tissue.

In the medulla oblongata fragments were identified typically lesions for scrapie: neuronal vacuolation, neuronal degeneration, loss of neurons, astrocytosis and spongious aspect of the grey matter neuropil. The vacuoles in different sizes were empty and clearly separated. The vacuoles have been intraneural, paraneural, perineural or non-associated with the perykaria (fig. 3). However, the absence of lesions is not an evidence of the absence of scrapie infection, as this can exist without either clinical signs or detectable morphological



changes (fig. 4) [15, 40]. Also, it is no direct correlation between the severity of clinical signs and pathological changes [5, 13]. Real-Time Polymerase Chain Reaction (RT-PCR) is a high sensitive technique and the presence of some contaminants can perturb the melting curves. The presence of inhibitors in the brain homogenate may influence the RT-PCR, that for confirming the accuracy of RT-PCR we used the sequencing technique. The sequencing technique is the main tool used to identify the allelic variant of the sheep PRNP gene [6, 14, 20, 22, 31]. The study was extended to exon 3 of the sheep PRNP gene where 3 sequence variants were identified, all of them with single nucleotide polymorphism. The sequencing analysis confirmed the complexity of sheep PRNP genetics previously described [1, 8, 22, 35]. The primers amplified a 402pb fragment (from p22556 to p22958) of the ovine prion protein gene (GenBank access number U67922). The gene fragment has 3 polymorphic codons: 136, 154 and 171. Figure 6 presents the nucleotide sequence of the PRNP gene variants, aligned by Bio Edit software (the ClustalW application). In addition to the known polymorphisms from GenBank, we detected one new amino acid change in sample 14580 half breed of Merinos ARQ/ARQ genotype. The single nucleotide polymorphism was at codon 185 (ATC-ACC), an Isoleucine-to-Threonine substitution. The last two variants show polymorphism at codons 143 (TBHB, female, 30 months, scrapie positive, genotype ARQ/ARQ) and 171 (HBM, female, 24 months, genotype ARH/ARQ) (fig 6, 7, and 8), respectively. A Histidine-to-Arginine substitution at codon 143 was previously described [26], with low frequency. The single nucleotide polymorphism at codon 171 (CAG-CGG), a Histidine-to-Glutamine substitution was also previously described with high frequency [37, 21].

Chanel 640

(a)

(c)

(e)

Chanel 705

(b)

(d)

(f)

Figure 5. The melting curves of scrapie-positive animals: (a), (b) genotype ARQ/ARQ (MMB No. 14580); (c), (d) genotype ARQ/ARQ (TBHB no. 14703); (e), (f) genotype ARH/ARQ (MMB No.15456).

Figure 6. The ovine PRNP gene polymorphism at 143, 171 and 185 codons. (ClustalW alignment with the GenBankU67922 sequence).



Figure 7. The ovine PRNP amino acid sequence polymorphism. (ClustalW alignment with the GenBankU67922 sequence).

Figure 8. PRNP gene polymorphisms of Romanian sheep ecotypes confirmed with scrapie: (a) His-Arg substitution at codon 143 (TBHB, female, 30 months, scrapie-positive, genotype ARQ/ARQ); (b) His-Gln substitution at codon 171 (HBM, female, 24 months, genotype ARH/ARQ); (c) Ile-Thr substitution in codon 185 (HBM, female, 20 months, genotype ARQ/ARQ) Conclusions The evaluation of genetic risk for scrapie in a traditional Romanian sheep farm, officially confirmed with scrapie, proved the highly variation of susceptibility between sheep breeds of the farm. The greatest variability within the PRNP gene was expressed in Tigae with Black Head Breed sheep (TBHB), harboring 12 PRNP genotypes. The number of rams susceptible and highly susceptible to scrapie was important (29%), this feature making difficult the genetic selection. All EIA and histopathological positive results were confirmed by IHC and immunoblotting performed in NRL-TSE (IDAH, Bucharest, Romania). These results support the quality of procedures and methods for scrapie screening used in Romanian counties. The sequencing analysis confirmed the complexity of sheep PRNP genetics. Sequence-based PRNP genotype method in 11 scrapie-positive sheep revealed three gene variants. In addition to the known polymorphisms of GenBank, it was detected a new single nucleotide polymorphism at codon 185 (ATC-ACC), an Isoleucine-to-Threonine substitution, and two frequent reported substitutions at codons 143 and 171, a Histidine-to-Arginine substitution and a Histidine-to-Glutamine substitution, respectively. The results of this study confirm the importance of sequence-based genotype investigations in the sheep-breeding programs. Also, data support the scrapie control and eradication programs based on the gradual elimination of haplotypes associated with scrapie susceptibility and the use in breeding of the ARR/ARR genotype rams. Acknowledgments The work of Otelea Maria Rodica was performed as part of the project POS-DRU/88/1.5/S/52614 “Doctoral Scholarships for high quality training for young researchers in the field of agronomy and veterinary medicine”, co-financed by European Social Fund with the Operational Program Human Resources Development 2007-2013, area of intervention: 1.5. Doctoral and Post-doctoral Programs to support the research.



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the scrapie genetic susceptibility of some sheep breeds in ... otelea rodica.pdf · note: our study...

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