1 Federal Research Centre for Virus Diseases of Animals, Institute for Novel and Emerging Infectious Diseases, Boddenblick 5a, 17493 Greifswald Insel Riems, Germany
2 Department of Animal Breeding and Genetics, Justus-Liebig-University Gießen, Ludwigstraße 21B, 35390 Gießen, Germany
Correspondence
Martin H. Groschup
martin.groschup{at}rie.bfav.de
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ABSTRACT |
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INTRODUCTION |
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The diagnostic methods currently applied to detect a TSE infection (BSE rapid tests as well as confirmatory methods) are based on the detection of PrPSc. In contrast to its cellular counterpart (PrPC), PrPSc is partially proteinase K (PK) resistant and forms scrapie-associated fibrils (SAFs) because of its high hydrophobicity (Diringer et al., 1983; Hope et al., 1986
; Oesch et al., 1985
; Lehmann & Harris, 1995
). The four commonly used rapid tests as well as the confirmatory methods [SAF immunoblot and immunohistochemistry (IHC)] that have been recommended by the Office International des Epizooties (OIE) apply polyclonal or monoclonal antibodies to detect PK-treated PrPSc accumulated in the brains of TSE-affected animals.
In April 2002, an obligatory large-scale rapid-testing programme on slaughtered and fallen sheep and goats was implemented for monitoring purposes in the European Union (EU) and has revealed considerable numbers of scrapie cases in many member states. Because of the theoretical risk of transmission of the BSE agent to sheep and goats, the eradication of scrapie has become a high priority in the EU. These two infections cannot be distinguished by clinical symptoms or common diagnostic methods alone. Unequivocal discrimination requires comparison of the biochemical properties of the PrPSc or strain typing by lesion-profile scoring, which is performed by mouse bioassay in three conventional mouse lines (Bruce et al., 1996; Fraser & Dickinson, 1968
). It is generally accepted that the susceptibility of sheep to scrapie is directly linked to particular allelic polymorphisms of PrP. Sheep carrying alleles encoding valine/arginine/glutamine (PrPVRQ) or alanine/arginine/glutamine (PrPARQ) at amino acid positions 136, 154 and 171 of PrP are highly susceptible, whereas alleles encoding alanine/arginine/arginine (PrPARR) seem to protect against this disease, particularly when homozygous (Goldmann et al., 1990
; Hunter, 1996
, 1997
; Hunter et al., 1997
). With one questioned exception (Ikeda et al., 1995
), scrapie has never been diagnosed in a PrPARR homozygous sheep. In the UK, France, The Netherlands and many other EU member states, large genotyping and breeding programmes have been started in order to increase the number of so-called scrapie-resistant sheep (Arnold et al., 2002
; Dawson et al., 1998
). In scrapie-affected sheep flocks, recent eradication strategies rely on the removal of sheep that are considered genetically susceptible and on the selective breeding of so-called scrapie-resistant animals.
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METHODS |
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Confirmatory testing: SAF immunoblot.
For preparation of SAFs, a 10 % (w/v) homogenate was prepared of 1 g brainstem material from the obex region in 0·01 M sodium phosphate buffer, pH 7·4, containing 10 % (w/v) sarcosine, 0·5 mM PMSF and 0·5 mM N-ethylmaleimide. After a preliminary centrifugation for 30 min at 20 000 g to pellet residual detritus, the supernatant was transferred into a new centrifuge tube and centrifuged for 135 min at 220 000 g. Pellets were resuspended in 3 ml 0·015 M Tris/HCl pH 7·4 and incubated for 15 min at 37 °C and twice the sample volume of 15 % potassium iodide/high-salt buffer containing 0·1 M sodium thiosulphate pentahydrate, 0·3 M N-lauroylsarcosine and 0·01 M Tris/HCl was added. Samples were incubated at 37 °C for a further 30 min. Samples were split into equal parts and 45 µg PK was added to one of the aliquots and incubated for 60 min at 37 °C. Afterwards, 4·5 ml 10 % potassium iodide/high-salt buffer was added to the digested and untreated aliquots. Finally, samples were centrifuged through a 20 % sucrose gradient for 60 min at 280 000 g. Pellets were resuspended in a sample buffer pH 6·8 containing 1 % (w/v) SDS, 25 mM Tris/HCl pH 7·4, 0·5 % mercaptoethanol and 0·001 % bromophenol blue, heat-denatured for 5 min at 95 °C and loaded on SDS-polyacrylamide gels containing 13 % bisacrylamide. After electrophoresis, proteins were transferred to a PVDF membrane in a semi-dry chamber. Membranes were blocked in I-Block (Tropix) for 30 min and incubated with the PrP-specific monoclonal detection antibody (mAb) L42 (specific for both PrPC and PrPSc) (Harmeyer et al., 1998) for 90 min at room temperature. Membranes were washed three times for 10 min with PBS containing 0·1 % Tween 20 and then incubated with a secondary antibody bound to alkaline phosphatase (goat anti-mouseAP; Dianova) for 60 min at room temperature. After washing, the chemiluminescence substrate CDP-Star (Tropix) was applied and incubated on the membrane for 5 min, before the light signals on the membrane were detected directly in a camera.
PrPSc detection by IHC.
Samples were processed as described previously (Hardt et al., 2000). Briefly, 3 mm sections of the obex region were fixed in 3·5 % sodium-buffered formalin (SBF) for at least 48 h. After a 60 min incubation in 98 % formic acid, samples were dehydrated automatically with pressure and vacuum at 35 °C through a series of ethanol solutions and embedded in paraffin blocks. Sections (3 µm) were then prepared and IHC staining with the PrP-specific mAb L42, binding to an epitope at aa 145163 of ovine PrP (Harmeyer et al., 1998
), or SAF 70, binding to an epitope at aa 142160 of hamster PrP (Demart et al., 1999
), was done in an automated stainer. This procedure included a pre-treatment for 15 min in 98 % formic acid followed by a 5 min incubation in tap water, 30 min in SBF and washing twice in PBS for 5 min before placing the slides into a Ventana Discovery autostainer. The automated staining protocol included a heat treatment at 95 °C for 12 min followed by a protease treatment for 12 min at 42 °C. After blocking the slides in 30 % goat serum, they were incubated with primary antibody for 20 min at room temperature. This was followed by a washing procedure, incubation for 2 min with biotinylated Ig (Ventana), washing again and then incubation with streptavidinhorseradish peroxidase for 8 min. Signals were visualized with the diaminobenzidine detection system and hydrogen peroxide. Finally, sections were counterstained with haematoxylin and blueing reagent. All reagents used in this protocol were supplied by Ventana. As controls, brain sections from the obex region and cerebellum from a TSE-negative sheep and from a sheep with classical scrapie were used.
Determination of PrP allele.
PrP alleles of the diseased sheep were determined by sequencing and by PCR-RFLP (Lühken et al., 2004). Briefly, genomic DNA was extracted from brain samples with a commercial kit (QiaAmp DNA kit) followed by PCR amplification. To generate templates for sequencing, primers 5'-TGCCACTGCTATACAGTCATT-3' (sense; nt 2217922199 of GenBank sequence U67922) and 5'-TGGTGGTGACTGTGTGTTGCT-3' (antisense; nt 2284122861) for amplification of a 682 bp fragment and primers 5'-AACCAACATGAAGCATGTGGC-3' (sense; nt 2260422624) and 5'-AAGCAAGAAATGAGACACCACC-3' (antisense; nt 2312723148) for amplification of a 544 bp fragment were used in reaction mixtures comprising, in a volume of 50 µl, approximately 200 ng genomic DNA, 20 pmol of each primer, 5 mM of each dNTP, 2·0 mM (682 bp fragment) or 3·0 mM (544 bp fragment) MgCl2 and 0·25 U Taq polymerase in onefold reaction buffer with the following PCR conditions: denaturation at 94 °C for 1·5 min, 40 amplification cycles of denaturation at 94 °C for 15 s, annealing at 59 °C (682 bp fragment) or 60 °C (544 bp fragment) for 20 s and extension at 72 °C for 45 s, followed by a final 5 min extension at 72 °C. The PCR fragments were used directly in sequencing reactions or restriction enzyme digestions for determination of the DNA codons at positions 136, 154 and 171 of the ovine PrP. For sequencing, each of the four PCR primers was reused, which resulted in sequences covering the complete coding region of exon 3 of ovine PRNP.
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RESULTS AND DISCUSSION |
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Following this diagnosis, all animals in the flock from which the affected sheep were derived were genotyped, and animals carrying no PrPARR allele or the PrPVRQ allele in the homozygous or the heterozygous form were culled. No other scrapie case was found by rapid testing (Bio-Rad Platelia) within this selected group. This may result at least partly from the fact that no PrPSc was detectable in the analysed lymphoid tissue of this animal, which reduces the risk of horizontal scrapie transmission within a flock.
In the course of retrospective genotyping of all German scrapie cases, DNA was prepared from brain tissue (three independent samples) and from masseter muscle of the scrapie-diagnosed animal and sequenced by RFLP analysis. Surprisingly, all tests consistently gave the PrP genotype of this animal to be PrPARR/ARR. Genotyping of this sheep was repeated by another laboratory and the PrPARR/ARR allele was again confirmed. Other than the amino acid substitution at codon 171 (QQ/RR), no further nucleotide variations were observed comparing the sequence obtained, covering the complete coding region of exon 3 of PRNP, with GenBank sequence U67922. This is the second time that a PrPARR/ARR genotype has been found in a scrapie case from Germany. A previous case, designated ARR I, which was also discovered retrospectively by genotyping, occurred in an 810-year-old female sheep that was sent for rendering, and was tested using the Bio-Rad Platelia rapid test as a routine sample. The only available brain-tissue sample from this case was heavily autolysed and of a paste-like consistency so that the obex region could scarcely be determined. The Platelia readings on the assumed obex sample were borderline reactive (absorbance in duplicate readings 0·231/0·554) in the first set of experiments and low reactive in the second set of experiments (absorbance in duplicate readings 0·426/0·492). However, no positive result was obtained when the Prionics Check Western or LIA or the Enfer rapid test were used. Accumulation of PrPSc was also found by SAF immunoblotting in two independent experiments (Fig. 1b). Again, the banding patterns did not match those of typical scrapie. IHC staining with mAbs L42 or SAF 70 on the assumed obex was negative, a result that could also have been because of the heavy autolysis or a sampling artefact. The suspicion of scrapie was eventually confirmed on the basis of the Bio-Rad Platelia and the SAF immunoblotting results. All sheep in the herd of origin were culled irrespective of their genotype, but no other sheep was found to be positive.
In this case, DNA purified from the reactive brain was sequenced and PrPARR/ARR allele was determined. To confirm this, another DNA preparation was genotyped, again in the same laboratory and in parallel in an independent laboratory. All investigations confirmed the first genotyping results of ARR homozygosity. Again, no nucleotide variations other than the amino acid substitution at codon 171 (QQ/RR) could be observed by comparing the sequence obtained with GenBank sequence U67922.
For both of the potential ARR scrapie cases, mouse bioassays have now been initiated to investigate the presence of inherent TSE infectivity. RIII, C57Bl, VM95 and ovine PrPC (PrPARQ)-overexpressing transgenic mice have been inoculated intracerebrally with cerebellar and/or obex homogenates (10 %, w/v). Results from these studies are not expected to be available before the middle of 2005.
It is well established that PrPARR allele homozygosity does not confer absolute resistance to TSE experimental challenge. PrPARR/ARR sheep experimentally exposed to BSE by intracerebral inoculation develop clinical signs of a TSE infection (Houston et al., 2003). PrPSc found in these animals displayed the same glycoform pattern as found in diseased sheep of the susceptible PrPARQ homozygous genotype. Moreover, cell-free conversion of PrPARR using typical scrapie as seed is possible, albeit at a much lower efficiency (Bossers et al., 2000
).
There has been no previous unquestioned report of a natural scrapie case in a PrPARR/ARR sheep, although a large number of diseased animals have been genotyped. However, the large majority of scrapie cases have been confirmed previously by IHC staining of PrPSc in the obex, which is considered the most reliable diagnostic marker of scrapie (Anonymous, 2004). Only in a few instances was SAF immunoblotting carried out, because this requires a larger quantity of sample and is more time-consuming. After the introduction of the scrapie-monitoring programme in the EU, in which obex samples are now being tested using rapid tests, atypical scrapie cases are being detected more frequently. It is intriguing to see that atypical scrapie cases have been uncovered recently in which PrPSc deposition at the level of the obex is faint or absent. As only the obex or brainstem is sampled for this programme, reassessing these cases by analysis of other brain areas is often impossible. Therefore, atypical scrapie cases may have been previously under-reported. However, it must be noted that the infectious nature of this novel scrapie type still has to be confirmed by transmission experiments. A similar situation may exist for yet unrecognized scrapie cases in PrPARR/ARR animals, which may display a distinct PrPSc deposition topology in the brain and a distinct biochemical glycotyping pattern. This novel phenotype may originate from a particular PrP genotype, from a peculiar scrapie strain or from the combination of the two.
It is interesting to see that SAF immunoblotting of the obex or cortex gave a positive result, like the Bio-Rad Platelia, while IHC staining for PrPSc was negative. It could be argued that IHC is much less sensitive at recognizing these particular cases. Moreover, our own results indicate that SAF preparation is in general more sensitive to dilution of positive samples than IHC staining (A. Buschmann and others, unpublished results). However, the heavy PrP staining in the cerebellum proves that this method is in principle equally suitable for detection of these cases.
Taken together, the findings reported here indicate that sheep homozygous for the PrPARR allele may not be fully resistant to natural scrapie infections and may exhibit diagnostic features that fit the most recently discovered atypical scrapie case definition. Atypical scrapie cases occur predominantly in sheep carrying a scrapie-resistance PrP allele in heterozygous or homozygous form. If transmission studies indeed show that the PrPSc depositions in these cases are infectious and that such infections are able to spread from sheep under natural conditions, these findings would question the large-scale sheep genotyping and scrapie-resistant breeding programmes that have been introduced in several EU member states over the last 5 years.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 28 January 2004;
accepted 11 May 2004.