Atypical scrapie cases in Germany and France are identified by discrepant reaction patterns in BSE rapid tests

Journal of Virological Methods 117 (2004) 27–36

Atypical scrapie cases in Germany and France are identified by discrepant reaction patterns in BSE rapid tests A. Buschmann a , A.-G. Biacabe b , U. Ziegler a , A. Bencsik b , J.-Y. Madec b , G. Erhardt c , G. Lühken c , T. Baron b , M.H. Groschup a,∗ a

c

Federal Research Centre for Virus Diseases of Animals, Institute for Novel and Emerging Infectious Diseases, Boddenblick 5a, 17493 Greifswald-Insel Riems, Germany b AFSSA-Lyon Unité “Virologie-ATNC”, 31 Avenue Tony Garnier, 69364 Lyon Cedex 07, France Department of Animal Breeding and Genetics, Justus-Liebig University Giessen, Ludwigstr. 21B, 35390 Giessen, Germany Received 27 August 2003; received in revised form 13 November 2003; accepted 18 November 2003

Abstract The intensified surveillance of scrapie in small ruminants in the European Union (EU) has resulted in a substantial increase of the number of diagnosed cases. Four rapid tests which have passed the EU evaluation for BSE testing of cattle are also recommended currently and used for the testing of small ruminants by the EU authorities. These tests include an indirect ELISA (cELISA), a colorimetric sandwich ELISA (sELISA I), a chemiluminescent sandwich ELISA (sELISA II), and a Western blot (WB). To this point, the majority of samples have been screened by using either sELISA I (predominantly in Germany) or WB (predominantly in France). In this study, it is shown that a number of the German and French scrapie cases show inconsistent results using rapid and confirmatory test methods. Forty-eight German sheep, 209 French sheep and 19 French goat transmissible spongiform encephalopathy (TSE) cases were tested. All cases were recognised by the sELISA I and either one of the confirmatory methods (scrapie-associated fibrils (SAF)-immunoblot or immunohistochemistry). Surprisingly, three rapid tests failed to detect a significant number of scrapie cases (29 in France and 24 in Germany). The possible reasons for these inconsistent reaction patterns of scrapie cases are discussed. Similar discrepancies have not been observed during rapid testing of cattle for BSE, the disease for which all diagnostic methods applied have been evaluated. © 2003 Elsevier B.V. All rights reserved. Keywords: Scrapie; Prion protein; Rapid test; Confirmatory method

1. Introduction Scrapie in sheep and goats is the longest known transmissible spongiform encephalopathy (TSE) and has first been described in the 18th century (McGowan, 1922). As no obvious clinical or epidemiological connection to human disease has been revealed to date, scrapie is considered non-pathogenic for humans, at least under natural conditions. However, an epidemic of bovine spongiform encephalopathy (BSE) in cattle emerged during the last two decades of the 20th century. Following this extensive exposure, a variant form of Creutzfeldt–Jakob disease in humans was discovered in 1996, which is linked directly to BSE, the bovine form of transmissible spongiform encephalopathy

∗

Corresponding author. Tel.: +49-38351-7163; fax: +49-38351-7191. E-mail address: [email protected] (M.H. Groschup).

0166-0934/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2003.11.017

(Will et al., 1996; Collinge et al., 1996; Bruce et al., 1997). Under experimental conditions, sheep are easily infected orally by the BSE agent and suffer from clinical signs indistinguishable from scrapie. In contrast to cattle, sheep carry abundant amounts of infectivity throughout most body tissues even early after infection (Foster et al., 1993, 2001). Although scrapie is a notifiable disease, the actual number of scrapie cases in the EU member states during the last years remained unclear. To identify the true incidence of this disease, EU regulations for an active surveillance have been implemented in the year 2002 (Moynagh and Schimmel, 1999). These regulations (EC regulation 999/ 2001 and amendments) require large scale TSE testing of small ruminants using EU approved rapid tests that have been successfully evaluated for BSE testing in cattle. Sample numbers that need to be tested in each member state were set according to the numbers of slaughtered and fallen animals in a member state and comprise up to 66,000 tests (for

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Germany and France). As a result of the obligatory active surveillance program, numbers of scrapie cases diagnosed increased considerably throughout Europe. For example, in Germany no or only single scrapie cases per year were reported until the year 2001, which added up to 12 cases in total (Junghans et al., 1998). In contrast, after implementation of the monitoring scheme, 35 cases were observed during 2002 followed by 13 cases during the first 7 months of 2003. Rapid tests are based on the detection of pathological prion protein (designated PrPSc ). In contrast to its cellular counterpart, PrPSc is partially proteinase K (PK) resistant, and due to its high hydrophobicity forms scrapie-associated fibrils (SAF) (Oesch et al., 1994; Lehmann and Harris, 1995). The four rapid tests used commonly well as the confirmatory methods that have been approved by the Office International des Epizooties (OIE; SAF-immunoblot and immunohistochemistry) apply polyclonal or monoclonal antibodies to detect the proteinase K-treated PrPSc that is accumulated in the brains of TSE-affected animals. As a natural BSE transmission to sheep cannot be excluded and would suggest a high risk of exposure for humans, the scientific and public attention towards scrapie and its discrimination from BSE has increased. Strain typing in mice is used to differentiate BSE and scrapie infection in sheep. It compares the incubation times and scores the pathological lesions that the transmitted sheep brain samples cause in inbred mouse lines (Fraser and Dickinson, 1968; Bruce et al., 1996). However, this method is time-consuming and the results may be ambiguous and therefore difficult to interpret. Recently, alternative methods for the strain differentiation have therefore been developed such as the interpretation of the molecular weight and the glycosylation pattern of the pathological and partially proteinase K-resistant form of the prion protein (PrPSc ) accumulated in the brain of the infected host (Hill et al., 1998; Hope et al., 1999; Kascsak et al., 1986; Kuczius et al., 1998; Sweeney et al., 2000; Baron et al., 1999; Groschup et al., 2000; Zanusso et al., 2003). Amino acid variations at positions 136 (valine (V) and alanine (A)), 154 (arginine (R) and histidine (H)) and 171 (glutamine (Q), arginine (R) and histidine (H)) of the ovine prion protein have been shown to influence the susceptibility of sheep to natural and experimental scrapie infections (Laplanche et al., 1993; Goldmann et al., 1994; Belt et al., 1995; O’Doherty et al., 2002). In general, the ovine alleles PrPVRQ and PrPARQ seem to be related to a higher susceptibility while the alleles PrPARR and PrPAHQ coincide with a low susceptibility to a scrapie infection. Therefore, the PrP alleles of the sheep TSE cases described here were also taken into consideration. To date, no independent comparative study has been performed to evaluate the rapid tests’ performances with sheep or goat scrapie samples. We report that a substantial number of scrapie cases which have been diagnosed using the approved confirmatory methods, e.g. SAF-immunoblotting and immunohistochemistry (OIE manual) could not be detected with three of the four rapid tests used currently.

2. Materials and methods 2.1. Sampling of sheep brains Brainstem tissue pieces (up to 10 g) were collected at abattoirs and rendering plants by sampling through the foramen magnum. Depending on their origin, sample quality varied from fresh to autolytic. 2.2. BSE rapid tests BSE rapid tests were performed following the manufacturers instructions. As each rapid test requires a specific homogenisation procedure and buffer developed solely for that test, homogenates that were prepared for one test method could not be exchanged with other tests. Moreover, due to lack of brainstem material, it was impossible in many cases to prepare a macerate of a larger amount of brainstem material for the preparation of comparable homogenates. In most cases, the initial rapid testing was repeated at the national reference laboratory using the original homogenate. For further testing using the same or another rapid test method, new homogenates from another piece of brain needed to be prepared. A colorimetric sandwich ELISA (sELISA I, Platelia, Biorad, Munich) was used for primary screening of most of the samples. Approximately 0.35 g brainstem tissues from the obex region were homogenised and digested with PK. After precipitation, samples were resolubilised and diluted before pipetting them on a microtitre plate that was coated with a PrP-specific monoclonal antibody. After intensive washing of the plate and subsequent incubation with the conjugate, the colorimetric signal was developed and the reaction stopped prior to measurement of the absorbance at 450 and 620 nm. The cut-off value to determine positive results was calculated by adding a fixed value of 0.21 to the mean optical density of the negative controls. For the indirect ELISA (cELISA, Enfer TSE kit, version 2.0, Enfer Scientific, Dublin), 0.5–1 g brainstem material was homogenised, a small amount of each sample was centrifuged to pellet residual detritus before samples were adsorbed on a microtitre plate and digested with PK. After intensive washing, samples were incubated with a PrP-specific polyclonal serum, washed again and incubated with conjugate, washed again before the chemiluminescence substrate was added. Emitted light units (LU) were measured and all samples with LU above 5.5 were considered as reactive. For the rapid Western blot (rapid WB, Prionics Check Testkit, Prionics, Zurich), 10% (w/v) homogenates were prepared from 0.5 g brainstem tissue and digested with PK for 40 min. The reaction was stopped by adding a protease inhibitor and samples were heat denatured. This was followed by sodium dodecylsulphate-polyacrylamide (SDS-PAGE), transfer to a PVDF membrane and incubation with a PrP-specific monoclonal antibody. After incubation with the conjugate, signals were visualised by

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chemiluminescence and detected using either film or a CCD camera system. Samples that gave signals of the molecular weight 15–29 kDa and showed the typical triple banding pattern, or at least the uppermost diglycosylated PrPSc band at 29 kDa were considered reactive. For the luminescence sandwich ELISA (sELISA II, LIA, Prionics), 10% (w/v) homogenates of 0.5 g brainstem tissue were digested with PK, incubated first with assay buffer and then with pre-incubation buffer before the PrP-specific monoclonal antibody was added to the samples. The samples were transferred to the detection microtitre plate that was coated with another PrP-specific monoclonal antibody and incubated at room temperature to allow the binding of PrP-detection antibody complexes to the microtitre plate. After intensive washing, a chemiluminescence substrate solution was added and light units were measured. The mean of eight negative control values multiplied by 10 was taken as negative control cut-off (NCC). The mean of all samples whose values lie under the NCC were multiplied by 10 to give the real sample cut-off (RSC). All samples with values higher than the RSC were considered as reactive. 2.3. Exchange of single rapid WB components All relevant test components were exchanged individually to examine their influence on the test results. To exchange the homogenisation buffer without having to prepare a fresh homogenate from the limited sample amount, 5% (w/v) brain homogenates were made by diluting the 10% (w/v) homogenates for the rapid test WB in a 1:2 ratio with either the same homogenisation buffer or with a 0.64 M sucrose buffer containing 1% (w/v) desoxycholate and 1% (w/v) Nonidet-P 40 (NP40; ICN, USA). In the following steps, the proteinase K solution supplied with the testkit was exchanged with 50 ␮g/ml PK from another supplier (Roche, Mannheim), or the primary antibody L42 was applied instead of the antibody included in the testkit, or the secondary antibody GAM-AP, Dianova was used to replace the conjugate from the testkit. We then replaced the solution as well as the primary and secondary antibodies at the same time (modified rapid Western blot). Finally, all relevant test components (homogenisation buffer, PK, primary and secondary antibodies) were replaced by the reagents used in the in-house BFAV rapid Western blot. 2.4. Proteinase K digestion of crude 10% brain homogenates (BFAV rapid Western blot) Some selected samples were also tested using a simple protease degradation protocol. Homogenates (10%, w/v) were prepared in a 0.32 M sucrose buffer containing 0.5% (w/v) deoxycholic acid sodium salt (DOC; Serva, Heidelberg) and 0.5% (w/v) NP40. These homogenates were incubated with 50 ␮g/ml PK (Roche, Mannheim) for 1 h at 37 ◦ C. Sample buffer containing mercaptoethanol was added and the reaction was stopped by adding 10 mM

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PMSF and heating to 95 ◦ C for 5 min. These samples were analysed in a SDS-PAGE as described further, using mab L42 that specifically binds to aa 144–166 of ruminant PrP (Harmeyer et al., 1998). 2.5. Confirmatory testing 2.5.1. SAF-immunoblot For preparation of the scrapie-associated fibrils, a 10% (w/v) homogenate in a 0.01 M sodium phosphate buffer (pH 7.4) containing 10% (w/v) sarcosine, 0.5 mM phenylmethylsulphonylfluoride and 0.5 mM N-ethylmaleimide was prepared with brainstem material from the obex region. The amount of material used depended on the initial rapid test result and on the amount of brainstem available (i.e. 2 g sample material or less was used if sELISA I absorbance was >1). After a first centrifugation for 30 min at 20,000 × g to pellet residual detritus, the supernatant was transferred into a new centrifuge tube and centrifuged for 2 h and 15 min at 220,000 × g. Pellets were resuspended in 3 ml of 0.015 M Tris (pH 7.4), incubated for 15 min at 37 ◦ C, and the double volume of 15% potassium iodide-high salt buffer containing 60 mM sodium pentahydrate, 10 mM Tris–HCl, and 1% N-lauroylsarcosine was added. Samples were incubated at 37 ◦ C for another 30 min and then split into equal parts before 45 ␮g PK was added to one of the aliquots and incubated for 1 h at 37 ◦ C. Afterwards, 4.5 ml of 10% potassium iodide-high salt buffer containing 60 mM sodium pentahydrate, 10 mM Tris–HCl, and 1% N-lauroylsarcosine was added to the digested and non-digested aliquots. Finally, samples were centrifuged through a gradient of 20% sucrose in 10% potassium iodide-high salt buffer for 1 h at 280,000 × g and the pellets were resuspended in a sample buffer (pH 6.8) containing 0.1 g/ml sodium dodecylsulphate, 25 mM Tris–HCl (pH 7.4), 0.5% mercaptoethanol and 0.001% bromphenol blue, heat denatured for 5 min at 95 ◦ C and loaded on SDS-PAGE gels containing 13% bis-acrylamide. After electrophoresis, proteins were transferred on a PVDF membrane in a semi-dry chamber. Membranes were then blocked in I-Block (Tropix, Bedford, USA) for 30 min and incubated with the PrP-specific monoclonal detection antibody (mab) P4 (Harmeyer et al., 1998) for 1 h and 30 min at room temperature. In cases with only a weak positive rapid test result, mab 6H4 was added to mab P4 as a second detection antibody. Membranes were washed three times for 10 min with phosphate buffered saline (PBS) containing 0.1% Tween 20 and then incubated with a secondary antibody bound to alkaline phosphatase (goat anti-mouse AP, Dianova) for 1 h 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 were detected in a camera. 2.5.2. Modified SAF-immunoblot Immunoblot analysis was carried out as described previously (Madec et al., 2000). Briefly, a 10% (w/v) homogenate

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in 5% (w/v) glucose was prepared from 0.35 g of tissue. Proteinase K was added to a concentration of 10 ␮g/ml and samples were incubated at 37 ◦ C for 1 h. N-Laurylsarcosyl was added to a final concentration of 10% (w/v). The samples were then centrifuged at 465,000 × g for 2 h over a 10% (w/v) sucrose cushion. The pellet was resuspended in sample buffer (4% (w/v) SDS, 2% (w/v) ␤-mercaptoethanol, 192 mM glycine, 25 mM Tris, 5% (w/v) sucrose) and subjected to immunoblot analysis as described earlier, using mab SAF 84 as a detection antibody. 2.5.3. Immunohistochemical PrPSc detection Samples were processed as described previously (Hardt et al., 2000). Briefly, 3 mm sections of the obex region were fixed in 3.5% natrium buffered formalin (NBF) for at least 48 h. After a 1 h incubation in 98% formic acid, samples were dehydrated automatically using pressure and vacuum at 35 ◦ C through a series of ethanol solutions and embedded in paraffin blocks. Sections (3 ␮m) were then prepared and immunohistochemical staining using the PrP-specific monoclonal antibody L42 was carried out in an automated stainer. This procedure included a pre-treatment for 15 min in 98% formic acid followed by an incubation for 5 min in tap water and for 30 min in NBF. The sections were then washed twice in PBS for 5 min before entering them into the autostainer. The automated staining protocol included a protease treatment for 12 min at 42 ◦ C. Signals were visualised using the Fast Red detection system. For weak positive samples, the same procedure was also applied using mab SAF 70, SAF 84 (Demart et al., 1999), and mab F89 (O’Rourke et al., 1998). French samples were analysed by immunohistochemistry as described previously (Debeer et al., 2001, 2002) using SAF 84 monoclonal antibody.

samples led to a substantial increase in the number of reported scrapie cases in sheep and goats from Germany and France. Since 2002, 48 cases of scrapie have been found in Germany and 228 in France by active surveillance (as of 31 July 2003). Thirty-eight of the German scrapie cases were initially detected by using the sELISA I, 9 by using the rapid WB, and 1 by immunohistochemistry, while 133 of the French cases (117 in sheep and 16 in goats) were initially detected by using the rapid WB and 95 (92 in sheep and 3 in goats) by using the sELISA I testing. All cases were confirmed by either one of the two standard diagnostic methods recommended by the OIE which are SAF-immunoblotting including a proteinase K treatment and ultracentrifugation, or immunohistochemistry. Both methods gave no PrPSc -specific false positive signals when brain material from a negative sheep was examined. SAF-immunoblotting of the sheep samples gave clear and unequivocal reactions with the typical PrPSc banding pattern (Fig. 1A). Some of

2.6. Determination of PrP genotypes PrP genotypes of the diseased sheep were determined by sequencing as described earlier (Junghans et al., 1998) and/or by PCR-RFLP. Briefly, genomic DNA was extracted from brain samples by using a commercial kit (QiaAmp DNA kit) followed by PCR amplification of the open reading frame of the PrP gene. The PCR fragments were directly used in sequencing reactions or restriction enzyme digestions for determination of the DNA codons at positions 136, 154, and 171 of the ovine PrP. 2.7. Statistics Fisher’s exact test of association was used for the non-parametric assessment of association between genotype and rapid WB results.

3. Results Similar to other observations after the introduction of mass screening for BSE, rapid testing of small ruminant

Fig. 1. OIE immunoblot, rapid Western blot, and immunohistochemistry for selected sheep samples. Note that samples A8G and A21G are not detected by rapid Western blot; sample A21G is also negative in immunohistochemistry. (A) Preparation of scrapie-associated fibrils following the OIE-approved protocol (for details see Section 2). Negative control sample and positive sample T22G were tested both with and without PK digestion, for other samples, only PK digestion could be performed due to low amount of available brain material. Mab P4 was applied as detection antibody. Amount of prepared brain material is given for each sample, three-fourths of each probe was loaded per lane. (B) Rapid Western blot of sheep samples as under (A). Test was performed following the manufacturers instructions. Mab 6H4 was used as detection antibody. (C) Immunohistochemistry for sheep samples as under (A) using mab L42 which specifically detects ruminant PrP. Signal detection using Fast Red system.

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Fig. 1. (Continued ).

the samples also displayed an additional band of a lower molecular mass around 12 kDa. When applied during diagnostic routine, immunohistochemistry revealed intra- and extraneuronal PrPSc deposition at the level of the obex and (where additional samples were available) at other sites with varying signal intensities (Fig. 1C and Table 1). We then retested the confirmed scrapie cases using the other available rapid tests. All eight German scrapie cases that had been initially detected by the rapid WB were also clearly recognised using the sELISA I rapid test. In contrast, 24 out of the 38 scrapie cases that had been detected by sELISA I with results varying between absorbance values of 0.5 and 2.3 were not detected by the rapid WB (Fig. 1B). These samples were therefore categorised as atypical scrapie cases. These samples were also negative when the sELISA II was used. Due to a lack of sample material, not all of these cases could be retested using the cELISA, but when 11 out of the 24 samples were analysed, all but 1 gave a negative result. In contrast, one out of the eight samples that had been initially recognised in the rapid WB and that could be retested using other methods, was also positive using the cELISA test (Table 1). As the cELISA has only been approved for Germany recently, no samples that had

first been detected by using this method were available for a comparison with other rapid tests. Atypical scrapie cases were also found in a number of the French samples (Table 2). From the 92 cases that had first been detected using the sELISA I test with absorbance values of up to 3.5 and that were confirmed by immunohistochemistry, 29 (28 sheep and 1 goat) samples were not recognised in the rapid WB. In contrast, samples that were initially detected using the rapid WB with signal intensities varying between weak and strong could be confirmed using the sELISA I and the other rapid tests whenever sufficient sample amounts were available. To follow-up the discrepancies between the various tests, PrPSc deposition patterns of all German cases with incongruent results were reassessed by immunohistochemical examination. Depending on the quality and freshness of the samples that had been sent to the national reference centre for confirmation, histological preparations could not in all cases be taken from the obex region. Although PrPSc staining of most scrapie cases was easily detectable, atypical cases also included three samples that were hardly recognised, and another six samples for which PrPSc deposition could not been detected at all. These results remained

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Table 1 Selected German scrapie cases displaying two different immunochemical PrPSc recognition patterns: while typical samples are confirmed positive with all methods (such as T3G, T5G, T6G, T10G, T11G, T12G, and T22G), atypical cases remain negative using the rapid WB, cELISA, and sELISA II test (such as A4G, A16G, and A18G) Sample

PrP genotype

sELISA Ia (cut-off range OD 0.22–0.29)

sELISA II (cut-off range OD 6459)

Rapid WB

cELISA (cut-off OD 5.5)

IHC

SAFimmunoblot

Material used for preparation (g)

T2G T5G T6G T10G T11G T12G T22G A2G A3G A4G A5G A6G A7G A8G A9G A10G A11G A12G A13G A14G A15G A16G A17G A18G A21G

ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ AHQ/AHQ ARQ/ARQ ARR/AHQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ AHQ/AHQ ARQ/ARQ AHQ/ARQ ARQ/ARQ AHQ/ARR AHQ/ARQ AHQ/AHQ ARQ/ARQ AHQ/AHQ AHQ/ARQ AHQ/ARQ

3.3 3.2 3.3 3.2/2.9 3.6 3.5 3.3/3.3 1.2 1.0 1.5/1.4 0.9 1.2/0.15 1.2/0.3 0.8 1.0/0.6 0.5 1.4 2.2 1.4 0.5 0.4/0.3 2.3 0.3/1.9 0.5 0.8/0.4

295789/126259 131465/156239 455848/866856 234241/280448 488483/470383 187268/210202 n.d. 68/99 213/205 108/103 268/285 125/132 145/146 116/132 104/110 195/186 131/132 111/98 121/128 109/104 90/90 110/118 133/125 91/92 n.d.

++ ++b ++b ++ ++b ++b ++ –b – – –b – – – – –b – – – –b – – – – –

n.d. 4720.3/5907.0 n.d. 1745.0/1685.3 1967.6/1901.1 n.d. n.d. n.d. 148.9/139.9 1.0/0.9 n.d. 3.5/6.5 n.d. n.d. n.d. n.d. 1.1/1.5 1.2/1.2 0.9/1.2 n.d. 0.9/1.2 0.6/0.7 n.d. 0.7/0.7 n.d.

+++ ++ +++ ++ + ++ ++ + ++ − − + + +++ + ++ − + + − − + +/− ++ −

++ ++ ++ ++ ++ ++ +++ + ++ ++ ++ ++ ++ ++ + ++ ++ + + + + n.d. + + +

1.5 1.5 1.5 1.5 1.0 1.0 1.0 1.5 2.0 1.5 2.0 1.5 1.5 2.0 1.5 2.0 1.5 1.0 1.5 1.5 2.0 – 2.0 2.0 1.5

n.d.: not determined. a Repetitive sELISA I readings obtained in the NRL are italicised. b Less than 0.5 g brain sample was used to prepare a 10% homogenate as the availability of these brain samples was limited.

Table 2 Reactivity patterns of selected French scrapie cases in sheep and one goat in sELISA I, rapid WB and immunohistochemistry Sample

PrP genotype

IHC (obex)

sELISA I (cut-off OD 0.23)

Rapid WB

S16F S1F S2F S3F S4F S5F S6F S7F S8F S9F (goat) S10F S11F S12F S13F S14F S15F S17F S18F S19F

ARQ/AHQ ARR/ARQ ARR/ARQ ARR/ARQ n.d. ARR/ARQ ARQ/ARQ ARR/ARQ n.d. – ARQ/ARQ n.d. ARQ/ARQ ARQ/ARQ n.d. n.d. n.d. n.d. n.d.

++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++

2.907 3.469 3.041 0.705 >3.500 0.925 >3.500 3.081 0.474 3.409 0.821 0.888 1.276 3.137 1.200 2.397 2.517 2.086 2.942

– – – – – – – – – – – – – – – – – – –

unchanged when different detection antibodies, such as SAF 70, SAF 84, and F89, were applied. To examine further the failure to detect a significant number of scrapie samples using the rapid WB, single reagents or procedures within the protocol were exchanged. Supplementation of the kit included homogenisation buffer with 0.32 M sucrose containing 0.5% (w/v) DOC and 0.5% (w/v) NP40 resulted in a minor increase of the signal strength of the pathological PrP. A similar slight increase of the detectable PrPSc signal was achieved when the homogenate was digested using 50 ␮g/ml proteinase K instead of the PK solution included in the kit with an undisclosed concentration. The same minor effects occurred when the in-house monoclonal antibody mab L42 (instead of mab 6H4 in the kit) or a different secondary antibody were used. However, when proteinase K and the first and secondary antibodies were exchanged at the same time, most of the deviant samples were recognised in the so modified WB assay (Fig. 2A and B). When samples were homogenised in 0.32 M sucrose (containing 0.5% (w/v) DOC and 0.5% (w/v) NP40) and proteinase K as well as the first and secondary antibodies were exchanged (referred to as BFAV rapid Western blot), the recognition of deviant samples was again improved (Fig. 2C).

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Fig. 2. Rapid Western blot of scrapie-affected sheep using different preparation and detection protocols (for details see Section 2). (A) Rapid WB kit (kit-included sample homogenisation buffer, digestion with kit-included proteinase K for 40 min at 48 ◦ C, signal detection using mab 6H4, kit-included goat anti-mouse conjugate). (B) modified rapid WB protocol where proteinase K, first antibody mab 6H4, and conjugate have been exchanged with 1 mg/ml proteinase K, mab L42, and GAM-AP. (C) BFAV rapid Western blot protocol where the sample homogenisation buffer, PK, blocking solution as well as first and secondary antibodies have been exchanged with 0.32 M Sucrose containing 0.5% DOC and 0.5%, 1 mg/ml proteinase K, mab L42 as first antibody and GAM-AP as secondary antibody. Note that samples A3G, A7G, and A10G are negative in the rapid Western blot protocol and positive using the modified rapid WB protocol and the BFAV rapid Western blot protocol.

To exclude any insufficient proteolytic degradation as a reason for the inconsistent rapid test results and to explore the PK sensitivity of atypical scrapie cases, different PK concentrations were applied to the samples. In our hands, 2.5 ␮g PK/ml were sufficient to digest reliably all PrPC from a negative sheep control sample and therefore abolish any banding signal in the SAF-immunoblot. On the other hand, a positive scrapie sample still gave a strong PrPSc signal even after exposure to 500 ␮g/ml PK, while the PrPSc signal of an atypical scrapie case was only clearly detectable after incubation with 50 ␮g/ml (Fig. 3). French scrapie cases were also tested using a modified SAF-immunoblotting protocol, in which the first ultracentrifugation step was omitted and the homogenate was treated directly with proteinase K. Clear PrPSc signals were seen in 66 samples initially detected using the sELISA I test as well as in 131 samples detected using the rapid WB. However, in 29 atypical samples no PrPSc banding pattern was

observed. Selected samples were subsequently tested by the original SAF-immunoblot and distinct PrPSc patterns were demonstrated (Fig. 4). The PrP genotypes of all German and 63 French scrapie cases (wherever suitable material was available) were determined. Without employing a full statistical analysis on relative allele frequencies, it seemed that atypical cases are not linked to one particular PrP genotype or allele but derive from a variety of genotypes: ARQ/ARQ, ARQ/AHQ, AHQ/AHQ, ARQ/ARR, and AHQ/ARR (in decreasing order of assumed scrapie susceptibilities). Fisher’s exact test revealed no significant association between a distinct PrP genotype and the result of rapid WB. A straight linkage between the non-uniform diagnostic recognition and genetic effects of a particular allele can therefore be excluded. However, it should be noted that 6 out of 10 deviant French and 10 out of 20 deviant German sheep carried an allele assumed to be linked to a higher resistance to scrapie (ARR or

Fig. 3. To compare the PK resistance of PrPSc derived from typical scrapie samples with positive results in all tests to that of samples with non-uniform results, we performed SAF preparations with PK concentrations from 2.5 to 500 ␮g/ml. While PrPC from an uninfected sheep control was completely digested by 2.5 ␮g/ml, PrPSc from a typical scrapie sample (T2G) still gave a strong signal after incubation with 500 ␮g/ml for 1 h at 37 ◦ C. PrPSc from an atypical scrapie sample (A24G) was almost completely digested after incubation with 250 ␮g/ml. L42 was used as a detection antibody.

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Fig. 4. Confirmation of six French scrapie cases by OIE approved SAF-immunoblotting according to the OIE approved protocol. Sample amounts of 0.7 g were purified and extracts loaded per lane. Mab P4 was used as a detection antibody and the light emission of CDP-Star was recorded using an imaging system. Note strong PrPSc banding patterns which were found in the atypical samples. As detection antibodies, a combination of mabs P4 and 6H4 was applied, conjugate GAM-AP was used.

AHQ) whereas such genotypes were found in only 4 out of 53 French and in no other German case that gave positive results in all tests. Similarly, incongruent results were found in sheep belonging to several breeds (e.g. Suffolk, Merino, Texel) so that a correlation to specific breeds may also be ruled out.

4. Discussion Since the spring of 2002, rapid tests are being used for active surveillance of scrapie in the national sheep herds of the EU member states. These tests have been approved by the European commission and they include a colorimetric sandwich ELISA, a luminescence sandwich ELISA, an indirect ELISA using chemiluminescence as well as a rapid Western blotting assay. According to EU legislation (EC regulation 999/2001 and amendments), rapid tests are only approved for screening brain samples. In case of a reactive result, the sample has to be examined in the national reference laboratory using one of the OIE approved confirmatory methods which are SAF-immunoblotting and immunohistochemistry. The introduction of this active surveillance programme in the EU using BSE rapid tests demonstrated that scrapie in small ruminants is much more prevalent than

had been previously estimated. Moreover, we found that not all scrapie cases are detected equally well by the four applied rapid tests as a significant portion of the samples were found positive with the sELISA I test but were not reactive with the rapid WB and sELISA II. The cELISA could only be used on a rather small number of samples, however, it must be assumed from the available results that it shows a performance similar to that of the rapid WB and sELISA II. Although some of the sELISA I results were weak positive, all cases that were initially detected using this method were confirmed by using OIE-recommended methods. It is therefore concluded that the sELISA I results are true positive, whereas the rapid WB, sELISA II and cELISA results of the same samples must be considered as false negative. This of course presupposes that the currently applied confirmatory methods do not produce any false positive results. All samples that were first detected by using the rapid WB also gave positive results using the other rapid test methods. This observation supports the hypothesis that the rapid WB fails to detect certain positive sheep. Regrettably, it was not feasible during this study to retest small ruminant field samples that had initially been negative in the rapid WB with the sELISA I test in order to check if any positive samples had been ignored during the first screening. Non-uniform rapid test results were reproducible when selected coded samples were exchanged between the German and French national TSE reference laboratories and the samples were repeatedly positive in the sELISA I. Furthermore, it should be emphasised that the reactivity using this test was generally high (more than four times the cut-off level in most cases), showing that negative results obtained with other methods cannot be explained by threshold levels of protease resistant prion protein in these particular samples alone. Brainstem samples collected in abattoirs and rendering plants do not always fulfill the desired diagnostic quality standards in terms of freshness and in sampling localisation. In the beginning, we therefore could not exclude the possibility that single incongruent test results were due to varying PrPSc concentrations in the brainstems. However, the large number of such samples and the variety of the sample histories argue against such artefacts. However, such effects may explain why a few cases were negative by immunohistochemistry, but positive by SAF-immunoblotting, a diagnostic method where PrPSc is concentrated from larger brain areas. These 53 atypical scrapie cases in France and Germany out of a set of 276 may represent a novel strain of this disease in the field. Similar observations have also been made by the Norwegian National TSE Reference Laboratory, where some sheep scrapie samples were not reactive using the rapid WB (Benestad et al., 2003). However, not all characteristics described for those Nor98 designated cases match the atypical scrapie cases reported here. In particular, all Nor98 cases display a strong signal at 12 kDa that seems to be absent in a number of the German and French cases. We therefore postulate that at least three different scrapie phenotypes

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(‘typical’ scrapie and two atypical strains) exist within the European sheep flock. To determine critical factors affecting the rapid WB detection of PrPSc , we undertook a series of exchange experiments. The detection of atypical cases was improved after replacing the rapid WB homogenisation buffer supplied within the testkit of which the composition is undisclosed by an in-house homogenisation buffer containing desoxycholate and NP40 as detergents. Attempts were also made to replace the proteinase K solution, the detection antibody (by mab L42 that is directed to the same epitope as mab 6H4), and the conjugate antibody. Although no single critical step was revealed that alone enabled the detection of samples, these modifications altogether led to positive results for almost all atypical samples. It became also evident that minor changes in the sample treatment may have major effects when the modified SAF-immunoblotting technique was applied: while some of the atypical samples were negative with this method in which the first ultracentrifugation step is omitted and PK digestion is performed prior to SAF preparation, the same samples became clearly positive when the other SAF preparation protocol was applied. However, the physiochemical characteristics of ovine PrPSc derived from different scrapie isolates that are the basis for the observed effects still need further research efforts. PrP molecules derived from animals affected with different TSE strains vary in their cleavage sites for proteinase K digestion and therefore display different molecular weights when analysed in immunoblot (Hill et al., 1998; Baron et al., 2000; Stack et al., 2002). For example, the N-terminus of PrP derived from BSE-affected sheep is digested further by proteinase K than PrP derived from scrapie-affected sheep and therefore leads to a residual PrP of a lower molecular mass. This effect has been proposed as a possible diagnostic marker to differentiate between BSE and scrapie infections in sheep. Our experiments showed that this variation in the PK cleavage site between scrapie and BSE in sheep has no impact on the performance of the rapid tests on ovine BSE PrPSc since all commercial rapid tests detect PrPSc derived from experimentally BSE infected sheep of the PrPARQ/ARQ genotype (data not shown). Moreover, WB and sELISA II use the same monoclonal antibody (mab 6H4; Korth et al., 1997) which binds to an epitope far away from the PK cleavage site (aa 144–148) and would therefore not be expected to react differently with scrapie- and BSE-derived PrPSc . The same is the case with the French immunoblot test that uses mab SAF 84 (Demart et al., 1999), binding to an epitope in the same region of the protein (aa 125–163). No information is available on the PrP epitopes that are targeted in the sELISA I and cELISA. TSE infectivity can only be confined reliably, to date, by transmission experiments to an appropriate host. As it cannot be ruled out completely at this stage that non-infectious PrPC may also form intracellular aggregates with increased protease resistance and hydrophobicity that may lead to false positive results in diagnostic tests, the level of infectivity of such atypical cases is currently being examined by in-

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oculation into RIII, C57B1 and VM95 mice. In case of a transmission to these mouse strains, the lesion profile scores will be determined and PrPSc will be analysed concerning its glycotype and PK resistance. The use of rapid tests for small ruminants was introduced by the EU Commission on the basis of their successful evaluation for BSE testing in cattle (Moynagh and Schimmel, 1999) in order to achieve an overview of the scrapie prevalence in the EU. Unfortunately, no independent evaluation has been performed in the EU to date to reveal the individual rapid tests performance on small ruminant scrapie cases. Therefore, their specificity and sensitivity for this use can only be estimated by the results of samples selected randomly that have been tested individually by the manufacturers. Inconsistencies in the ability of rapid tests to identify positive cases would question the current efforts to intensify and standardise the scrapie surveillance in the EU member states. Our data show that the actual numbers of scrapie cases and the prevalence of scrapie may be seriously underestimated in countries where rapid tests that may produce false-negative results are used. In the German epidemiosurveillance scheme for scrapie, the sELISA I is applied for testing of more than 80% of the samples, while in France 60% of the samples are tested with the rapid WB. Therefore, it must be accepted that the current EU-wide epidemiosurveillance programme can only give a general impression of the scrapie situation but may miss on average up to 12% of the true number of German scrapie cases and up to 16% of the French cases (estimated numbers take into account the applied test methods and the numbers of atypical cases since 2002). This must be kept in mind when scrapie prevalence data obtained by BSE rapid testing are interpreted.

Acknowledgements We wish to acknowledge Matthias Kramer and Sandra Göbel (FRCVDA-Wusterhausen, Germany) and Didier Calavas (AFSSA-Lyon, France) for epidemiological data, Bertrand Bed’hom (LABOGENA, France) for genotype analysis of French scrapie cases and J. Grassi (C.E.A.-Saclay, France) for supply of SAF 70 and SAF 84 antibody. This work was partly funded by the German Ministry of Consumer Protection, Nutrition and Agriculture (BMVEL).

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