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Journal of General Virology (2009), 90, 764–768

Short Communication

DOI 10.1099/vir.0.005983-0

Three serial passages of bovine spongiform encephalopathy in sheep do not significantly affect discriminatory test results Michael Stack,1 Lorenzo Gonza´lez,2 Martin Jeffrey,2 Stuart Martin,2 Colin Macaldowie,3 Melanie Chaplin,1 Jemma Thorne,1 Robin Sayers,1 Linda Davis,1 Jason Bramwell,1 Steve Grimmer1 and Sue Bellworthy1

Correspondence

1

Michael Stack

2

[email protected]

Veterinary Laboratories Agency (VLA), Woodham Lane, Addlestone, Surrey, UK VLA, Pentlands Science Park, Bush Loan, Penicuik, Midlothian, Scotland, UK

3

Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian, Scotland, UK

Received 28 July 2008 Accepted 17 November 2008

During the 1980s, bovine spongiform encephalopathy (BSE)-contaminated meat and bonemeal were probably fed to sheep, raising concerns that BSE may have been transmitted to sheep in the UK. The human disease, variant Creutzfeldt–Jakob disease, arose during the BSE epidemic, and oral exposure of humans to BSE-infected tissues has been implicated in its aetiology. The concern is that sheep BSE could provide another source of BSE exposure to humans via sheep products. Two immunological techniques, Western immunoblotting (WB) and immunohistochemistry (IHC), have been developed to distinguish scrapie from cases of experimental sheep BSE by the characteristics of their respective abnormal, disease-associated prion proteins (PrPd). This study compares the WB and IHC characteristics of PrPd from brains of primary, secondary and tertiary experimental ovine BSE cases with those of cattle BSE and natural sheep scrapie. Discrimination between experimental sheep BSE and scrapie remained possible by both methods, regardless of the route of challenge.

Scrapie, the ovine disease within the transmissible spongiform encephalopathy (TSE) group, has been endemic in the UK flock for .250 years. It is thought to be caused by an infectious prion protein (Prusiner, 1982) and is the most likely origin of bovine spongiform encephalopathy (BSE) (Wilesmith et al., 1991). A new form of human prion disease, variant Creutzfeldt–Jakob disease, developed in the wake of the BSE epidemic, and exposure of humans to BSE-infected tissues has been implicated in its aetiology (Will et al., 1996). The ban on the feeding of animalderived protein to ruminants and the apparent lack of vertical transmission of BSE have led to a decline in the incidence within the UK herd and, therefore, to a reduced risk for humans. However, sheep exposed experimentally by oral challenge with as little as 0.5 g BSE brain material contract infection and develop clinical disease (Foster et al., 1993). During the 1980s, BSE-contaminated meat and bonemeal were also fed to sheep, raising concerns that BSE may have been transmitted to, and be recycling within, the national flock, thus providing a secondary source of BSE exposure to humans via sheep products. This recycling scenario could arise as a result of natural vertical and Supplementary methods, references and figures are available with the online version of this paper.

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horizontal transmission of TSEs within flocks, dependent upon their genetics (reviewed by Baylis et al., 2002). Most TSEs are characterized by the accumulation of abnormal prion protein (PrPd) in the brain and extraneural tissues. Synthetic forms of this protein may transmit disease phenotypes without utilizing a nucleic acid genome by acting as a template, binding and changing the conformation of the normal cellular protein (PrPC) to replicates of PrPd (Legname et al., 2004). Although both have the same amino acid sequence, PrPd, unlike PrPC, is relatively resistant to proteolysis (PrPres) and insoluble in mild detergents. The traditional method available for typing BSE in sheep has been to infect inbred strains of laboratory mice experimentally and compare their relative survival times and brain histopathology (Bruce et al., 1997). Initial development of more rapid discriminatory techniques, based on differences in the molecular profile of PrPd by Western immunoblotting (WB) techniques (Hill et al., 1998; Hope et al., 1999), was followed by the development of enhanced discriminatory immunohistochemical (IHC) and WB techniques. These exploit the phenotype-dependent properties of PrPd to discriminate between experimental BSE in sheep and natural scrapie, by the use of two or more monoclonal antibodies (mAbs) for

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Printed in Great Britain

Effect of serial passages of BSE in sheep

robust discrimination (Jeffrey et al., 2001a; Stack et al., 2002). The principles behind the IHC and WB discriminatory tests and references to previous research on the use of such techniques are provided in the supplementary information, available in JGV Online. In order to validate discriminatory techniques, it is critical to know whether the differences between PrPd of primarypassaged experimental BSE and natural scrapie in sheep are retained after serial passage of the BSE agent. This paper describes the results obtained with these discriminatory WB and IHC techniques in samples from sheep primarily infected with bovine BSE, and from secondary- and tertiary-passaged sheep BSE, in comparison with the results obtained for natural scrapie and bovine BSE cases. Primary experimental infections were done by oral dosage of 5 g from a brain pool prepared from BSE in cattle that showed clinical signs and were confirmed by using statutory diagnostic tests. Eight sheep from scrapie-free sources, three Romney and five Suffolk breed, all of the PrPARQ/ARQ genotype, were dosed at 6 months of age. Two groups of sheep were available for the study of secondary-passaged BSE. The first comprised eight PrPARQ/ARQ sheep dosed orally with pooled brainstems from Romney sheep that had succumbed to cattle BSE primary challenge. The second group consisted of 12 PrPARQ/ARQ Romney sheep that developed clinical disease after intracerebral (i.c.) challenge with 0.5 ml of a 1023 (n55), 1024 (n55) or 1025 (n52) dilution of a sheep-brain homogenate. This was made from the brainstems of three clinically sick Romney sheep (PrPAHQ/AHQ genotype) previously dosed with a BSE cattle-brain homogenate (Jeffrey et al., 2001b). Tertiary experimental infections of PrPARQ/ARQ Romney breed sheep from scrapie-free sources were done by oral dosage of 5 g from a brain pool prepared from clinically affected sheep of the second oral BSE passage. A flow chart of donors and recipients is provided in Fig. 1. More details of the control animals used and the individual incubation periods are shown in the supplementary information, available in JGV Online. PrPres in ovine brain was characterized by SDS-PAGE/WB by using mAbs 6H4 and P4 as described previously (Stack et al., 2002). All samples, including controls, were taken from the caudal medulla brain region. Molecular masses, glycoform ratios and mAb 6H4 : mAb P4 signal ratios for the WB bands were measured as described previously (Stack et al., 2002, 2006; Nonno et al., 2003). Initial statistical analyses of variance of molecular masses of WB bands were done by using a main-effects model with terms for gel and sample (general linear models procedure in the STATISTICA software package). Several specific hypotheses were tested by t-tests using contrasts (linear combinations) of the sample means. For IHC, brains were fixed in 10 % neutral phosphatebuffered formalin, trimmed, post-fixed and embedded http://vir.sgmjournals.org

Fig. 1. Flow chart of donors and recipients (pools were all Romneys).

according to standard procedures. Tissue sections, 5 mm thick, were obtained from blocks containing the obex. An IHC protocol for the detection of PrPd was applied as described previously (Gonza´lez et al., 2002). The full range of antibodies used is described in the supplementary information, available in JGV Online. When WB was carried out using mAb 6H4, the cattle BSE and natural sheep scrapie positive-control samples and all of the sheep BSE samples gave a characteristic proteinbanding pattern corresponding to the three glycoforms of PrPres. The diglycosylated form (top band) showed the strongest signal, followed by the monoglycosylated form (middle band) and the unglycosylated form (lower band), which showed the weakest signal. A representative blot (Supplementary Fig. S1) and the molecular masses obtained (Supplementary Fig. S2) are available in JGV Online. Table 1 shows the statistical analysis of differences in molecular mass of the unglycosylated protein band between the different animal groups. Regardless of route of infection and number of passages, all sheep BSE groups showed an unglycosylated protein band of statistically significantly lower molecular mass than that of natural scrapie samples, but similar to that of cattle BSE samples, with the exception of primary and secondary i.c. passage BSE, which produced a band of statistically significantly lower molecular mass. Within secondary-passaged BSE, samples from orally dosed sheep showed statistically significantly higher molecular mass values for the unglycosylated band than those from i.c.-injected sheep. The glycoform-ratio results are shown in Supplementary Fig. S3 (available in JGV Online) and are based on the percentage signal obtained for the diglycosylated band plotted against that obtained for the monoglycosylated band. Due to the large SEM values obtained, this

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M. Stack and others

Table 1. Statistical analysis of the differences between the mean molecular masses (MM) of the unglycosylated WB bands obtained from all samples Abbreviations: NScr, natural scrapie control; cBSE, cattle BSE control; sBSE 1st, primary inoculation of cBSE in sheep by the oral route; sBSE 2nd, secondary passage of sBSE 1st in sheep by the intracerebral (i.c.) or oral (Or) route; sBSE 3rd, tertiary passage of sheep BSE by the oral route. Statistical differences between groups under comparison are shown as P-values; values in bold type are considered not statistically significant (P.0.05).

Mean MM (SEM) (kDa) Statistical difference (P-value) NScr cBSE sBSE 1st sBSE 2nd i.c. sBSE 2nd Or

NScr

cBSE

sBSE 1st

sBSE 2nd i.c.

sBSE 2nd Or

sBSE 3rd

19.55 (0.08)

18.72 (0.08)

18.25 (0.07)

18.18 (0.06)

18.64 (0.10)

18.67 (0.17)

– – – – –

,0.001 – – – –

,0.001 ,0.001 – – –

,0.001 ,0.001 0.461 – –

discriminatory criterion has previously been shown to be the least robust of the discriminatory criteria for this technique (Stack et al., 2002, 2006). Although the scrapie control sample could be discriminated from all of the BSE in sheep samples, the analysis could not discriminate between cattle BSE and scrapie. By using mAb P4 on the same samples, the cattle BSE control could not be detected at all, and all primary-dosed, secondary- and tertiary-passaged sheep had a greatly reduced reactivity to this mAb. Only the sheep scrapie control gave a strong signal with mAb P4. For 6H4 : P4 ratios, the total amount of PrPres signal emitted from all three protein bands was calculated and compared with that of a control scrapie sample, whose value was set at 1; the ratio for the unknown samples was then calculated relative to this control sample. Any ratio below 2 : 1 (total signal intensities for 6H4 : P4) was considered to be scrapie and any ratio above this was considered to be of BSE origin (Nonno et al., 2003; Stack et al., 2006). The results are shown in Fig. 2. All ratios in sheep BSE samples were above the cut-off value for the scrapie control, so discrimination was possible irrespective of the number of times that the BSE had been passaged or the route of infection. However, it can be observed that this ratio decreases as the number of passages increases. For IHC, antibodies raised to the C terminus (R145) and to the globular domain (6H4) of PrP gave strong immunolabelling of the brain sections examined, with intense and widespread neuropil (extracellular) and intraneuronal labelling. Intraneuronal PrPd was often most conspicuous in the lateral cuneate and olivary nuclei, although it was present in all neuronal nuclei to a greater or lesser extent. Intense granular intramicroglial labelling was also observed throughout the obex with the C-terminal and globulardomain antibodies. Although the remaining three antibodies tested (P4, 521 and 505), all of which recognize the upstream segment of the flexible tail of the PrP molecule, demonstrated extracellular, neuropil-associated labelling to 766

,0.001 0.506 0.003 0.001 –

,0.001 0.763 0.029 0.013 0.880

varying intensity, only the 505 antibody was able to demonstrate intraglial labelling. The P4 antibody did not show intraneuronal labelling in any of the neuronal nuclei examined, and antibody 521 recognized some intraneuronal, but not intramicroglial, PrPd. These immunolabelling features were the same regardless of the route of infection and the number of sheep passages of the inocula, and were also indistinguishable from those described previously for sheep challenged i.c. with cattle BSE (Gonza´lez et al., 2005; Martin et al., 2005). The differences are illustrated in two immunolabelled sections, shown in Supplementary Fig. S4 (available in JGV Online). In this study, although the IHC and WB results indicated that the serially passaged brain tissue retained the classical

Fig. 2. Antibody-signal ratios for the ovine scrapie sample (Ov scrapie), the bovine BSE positive control (Bov BSE +ve) and the BSE in sheep samples. The primary oral-inoculated BSE in sheep (16) gives the highest ratio of mAb 6H4 : P4 reactivity and this ratio decreases gradually in the secondary-passaged oral (26 oral) and secondary-passaged i.c. (26 i.c.) challenge. The ratio for the tertiary oral-passaged BSE in sheep (36) stays the same as that obtained in the 26 i.c. sheep. Numbers in parentheses are the total number of individual samples tested; controls were repeated measurements from single samples. There is no signal for the Bov BSE +ve with mAb P4 (normalized value of 100); the value has been adjusted to 24 to fit the graph scale. The dashed line indicates the classical scrapie cut-off point of 2.

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Journal of General Virology 90

Effect of serial passages of BSE in sheep

BSE-specific properties, it was apparent with all three WB analyses of results that it is possible that, on further experimental passage, a stage will be reached where discrimination may not be possible, due to the trend shown of a gradual change of profile for BSE in sheep to that found for natural scrapie cases. It is therefore important to continue the experimental passages to ascertain whether this will actually occur, and to study our experimental BSE in sheep flocks for horizontal or further vertical transmission and whether a possible natural change in molecular profile occurs. Although no cases of BSE have been detected in British sheep screened thus far (Stack et al., 2006), the confirmed case of BSE in a French goat (Eloit et al., 2005) emphasizes the importance of continued monitoring of small ruminants. It is now known that experimental BSE in sheep is clinically similar to natural scrapie and that, in the genotypes inoculated, non-neural tissues are also affected, which is not the case for BSE in cattle. If BSE has been transmitted to sheep in the UK, it may have been sustained over several generations by vertical and horizontal transmission within flocks, as is thought to be the case with scrapie infections. Preliminary results have shown natural transmission of BSE from dam to lamb, giving a BSE WB profile (Bellworthy et al., 2005). It has also been reported that the BSE WB and IHC profile is retained after a second passage in sheep by blood transfusion (Hunter et al., 2002; Siso´ et al., 2006), suggesting that changes in the molecular profile are not due to the route of infection. In this respect, it is worth pointing out that the WB differences found between oral and i.c. second-passage BSE in this study may not be due to the route of challenge, as the inocula used were also from different sources. It has been established that many different TSE strains exist and that, in experimental situations, their properties can sometimes, but not always, change after passage in a new host or in a new host with a PrP genotype different from that of the donor. Sometimes change does not occur until several passages have been performed (Bruce & Dickinson, 1987). Therefore, we do not know whether significant changes in the WB and IHC profiles may occur during further natural passages of the BSE agent between sheep, which would make it more difficult to differentiate it from scrapie by using our present methodology. This study deals with only one PrP genotype, two sheep breeds and two routes of inoculation, and any extrapolation should be careful. With our present knowledge of strain typing, it is difficult to interpret whether differences in PrP conformations are directly responsible for differences in molecular profiles after proteinase K digestion. If this information is transmitted directly from one molecule to another, it would infer that genetic information is being transmitted by this route and that changes would be genotypic in origin. However, if this is not the mechanism by which genetic information of TSE strains is transmitted, then the differences between PrPd would be phenotypic, involving other possible factors, including environment and genetics. http://vir.sgmjournals.org

Acknowledgements This study was funded by DEFRA. The authors are indebted to the Animal Services Unit at VLA and staff at the Moredun Research Institute for their husbandry, and to the post-mortem teams at the VLA Regional Laboratories and Neuropathology at Weybridge. Thanks also go to Jan Langeveld at CIDC-Lelystad, the Netherlands, for antibodies 521 and 505, and to Dr Danny Matthews and Dr James Hope at VLA for their critical comments on the manuscript.

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