BBSRC Institute for Animal Health, Neuropathogenesis Unit, Kings Buildings, West Mains Road, Edinburgh EH9 3JF, UK1
Author for correspondence: James Foster. Fax +44 131 668 3872. e-mail Jim.Foster{at}BBSRC.ac.uk
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Abstract |
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Introduction |
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Immunocytochemical methods for detection of abnormal deposits of PrP usually rely on antibodies which recognize both PrPC and PrPSc; however, the methodology can ensure that all that is visualized is PrPSc (Foster et al., 1996 ). In sheep, this disease-associated PrP has been observed using immunocytochemistry (icc) of fixed sections from brain and lymphoreticular tissue in animals with natural scrapie (Allsop et al., 1988
; McBride et al., 1988
; Miller et al., 1993
, 1994
; van Keulen et al., 1995
, 1996
; Schreuder et al., 1996
). Experimental studies in sheep challenged subcutaneously with two experimental sources of scrapie showed contrasting results. The SSBP/1 (Scrapie Sheep Brain Pool) source of scrapie produced very much lower levels of disease-associated PrP immunolocalization and scarce or no vacuolation in the brains of terminally affected sheep compared with the ME7 experimental scrapie strain and also the natural disease (Foster et al., 1996
).
Goats following intracerebral challenge with several different TSE sources (Foster & Dickinson, 1988 ; Foster et al., 1999
) develop disease accompanied by extensive vacuolar degeneration, especially in the thalamic nuclei. Goats are susceptible to experimental TSE challenge with incubation time controlled via polymorphisms of the PrP gene (Goldmann et al., 1996
). They have a higher incidence of disease following inoculation with sheep scrapie than do mice (especially for CH1641 and SSBP/1) and scrapie is a natural disease of goats, which makes them a most suitable model to examine TSE biological characteristics. It is also theoretically possible that, like cattle, UK goats could have become infected by BSE through contaminated feed and, therefore, the characteristics of BSE in goats are of interest.
In the present study, brain sections from goats which had been challenged intracerebrally with the SSBP/1 and CH1641 scrapie sources and with BSE, were examined for colocalization of vacuolation and the presence of disease-associated PrP using icc. Western blotting analysis of PrPSc (treated with proteinase K) from the brains of SSBP/1- and BSE-infected goats was also carried out to compare the PrPSc protein banding patterns resulting from different ratios of the mono-, di- and unglycosylated forms of the protein (glycoforms) (Hope et al., 1999 ).
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Methods |
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Sources of inoculum.
The following TSE sources were used in this study. SSBP/1 originated as an homogenate of three natural scrapie brains that were subsequently passaged mostly through NPU Cheviot sheep (Dickinson et al., 1989 ) with incubation times controlled by PrP genotype at codons 136, 154 and 171 (Goldmann et al., 1994
). SSBP/1 also produces disease in goats, although its linkage to PrP genotype has not been established (Goldmann et al., 1996
). For this study, eight goats challenged with SSBP/1 were examined.
The CH1641 isolate was recovered from a natural scrapie case in the NPU Cheviot sheep flock and generates distinctive incubation periods (Foster & Dickinson, 1988 ) related to PrP genotype at codon 171 (Goldmann et al., 1994
). CH1641 produces disease in goats related to genotype of the PrP gene at codon 142 (Goldmann et al., 1996
). Included in this study were three goats challenged with CH1641.
BSE infectivity was in the form of an homogenate of four terminally BSE-affected cattle brains. Diagnosis of all four cases was confirmed by histopathology and the transmission to mice from each BSE brain produced a pattern of disease which was characteristic of BSE (Fraser et al., 1992 ; Bruce et al., 1994
). BSE has been successfully transmitted to goats by experimental challenge (Foster et al., 1993
) with incubation periods related to PrP codon 142 genotype (Goldmann et al., 1996
). This study included six goats which had been challenged with BSE as part of an embryo transfer study (Foster et al., 1999
) and two which had been used for strain-typing BSE (Foster et al., 1993
).
Histology and immunocytochemistry.
Brain recovered at post-mortem was fixed by immersion in formol saline and processed according to a standard protocol (Sternberger et al., 1970 ). Paraffin sections (6 µm) were cut coronally to include the cuneate and dorsal vagus nuclei, pontine nuclei, mesencephalon, diencephalon, basal ganglia, parietal and frontal cortex. Sections for histology were stained with haematoxylin and eosin (H&E).
Sections for immunohistology were pre-treated with 98% formic acid for 5 min, and for 20 min with 0·1% trypsin at 37 °C in TrisHCl buffer pH 7·6, to enhance PrP immunolabelling. They were immunostained using the indirect two-step method with the primary antibody as a mouse monoclonal raised against either ovine recombinant PrP protein N-terminal sequence (FH11 at 1:3; Foster et al., 1996 ) or bovine recombinant PrP protein (BG4 at 1:3; Foster et al., 1999
) and the secondary antibody conjugated to peroxidase. The polyclonal antibodies 1A8 and 1B3 were raised in rabbits to mouse-passaged ME7 PrP, and used for comparative immunostaining (1:200; Jeffrey et al., 1994
). The antigenic amino acid sequences of the PrP protein with which both FH11 and BG4 react are numbered 4757 and 8999 (C. Birkett, personal communication), while those for 1A8 are 89114, 114131, 140182 and 187234. For 1B3 the sequences are 1436, 83102, 119139 and 188212 (Langeveld et al., 1994
).
Diaminobenzidine (brown) or aminoethylcarbazole (red) were used as substrates and all washes were performed with PBS buffer containing 0·2% BSA. Because no proteinase K is used in this method, positive staining is referred to as disease-associated PrP. In effect virtually all PrPC is removed during tissue processing and following the pre-treatments of formic acid and trypsin, which means that all immunostaining is really PrPSc.
The scoring system used to evaluate the intensity of both vacuolation and PrP immunostaining with icc is a previously reported subjective assessment by one of the authors (Foster et al., 1996 ) and is based on the range 0 to 5, where 5 is heavily vacuolated or stained (see Fig. 1).
PrP protein extraction and Western blotting.
At post-mortem, tissues for PrP protein extraction were flash frozen in liquid nitrogen and stored at -70 °C until required. No frozen tissue was collected from CH1641 cases because these challenge experiments culminated in the early 1980s, prior to the realization that tissue samples would be needed for PrP detection. PrPC and PrPSc protein was analysed by Western blotting, performed following the methods of Hope et al. (1986)
, and the rabbit polyclonal antibody 1B3 was used for immunolabelling PrPSc. Each track of the blotting gel was loaded with between 0·1 and 0·15 g of tissue from either SSBP/1 or BSE brain material.
Genotypes.
PrP genotypes generated as reported previously (Goldmann et al., 1994 , 1996
, 1998
) use the one-letter amino acid code: V, valine; A, alanine; Q, glutamine; R, arginine; I, isoleucine; M, methionine; G, glycine; W, tryptophan, with subscripts giving the codon numbers. The octapeptide repeat polymorphism is indicated by repeat numbers, 5 or 3. All goats were of repeat genotype 5/5 and were WW102 unless stated otherwise.
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Results |
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Infectivity was confirmed in the brain (thalamus) of one of these SSBP/1-infected goats (incubation of 596 days) when transmission was achieved by bioassay via subcutaneous inoculation into two NPU Cheviot sheep of PrP genotype VA136 QR171 with incubation periods of 370 and 473 days. These incubations are well within expected limits for this sheep PrP genotype challenged with SSBP/1 (Goldmann et al., 1994 ) and indicate, therefore, that infectivity levels in the brain of this donor goat are equivalent to those in terminally affected SSBP/1-challenged sheep. VA136 QR171 genotype sheep are also known not to develop natural scrapie in the NPU flock (Hunter et al., 1996
) and so the transmissions are genuine. The vacuolation profiles for both bioassay sheep in the medulla oblongata showed almost no signs of degeneration and were typical of SSBP/1 in sheep (Foster et al., 1996
). The 473 day bioassay case, however, exhibited slightly elevated levels of vacuolation in thalamic and cortical regions.
Detection of PrP by immunocytochemistry
Immunocytochemistry using the monoclonal antibodies BG4 and FH11 or the polyclonal antibodies 1A8 and 1B3 produced no or very little PrP immunostaining throughout the brains of goats inoculated with SSBP/1 or CH1641, even in areas where vacuolation was most intensive (Fig. 1b; Table 1). Four from eight goats challenged with SSBP/1 did exhibit a very mild, extracellular punctate staining in the thalamus; however, this was not related to PrP genotype (Fig. 1b). In one of the latter cases immunostaining was quite widespread, but light, throughout the thalamic nuclei, while the other three cases showed only localized focal staining. In the remaining four goats, disease-associated PrP either could not be recognized or was present as a few grains in each of the brain areas scrutinized. Only one of the three goats inoculated with CH1641 demonstrated any PrP immunostaining. This was restricted to a few grains of peri-neuronal decoration in the thalamus and was not related to PrP genotype or incubation period (Fig. 1f). In contrast, goats challenged with BSE exhibited (with antibody BG4) much stronger disease-associated PrP immunostaining, which was prominent in the thalamic (Fig. 1d; Table 1
) and hypothalamic nuclei (Fig. 1h; Table 1
), and in the basal ganglia.
Disease-associated PrP was also detected by icc in some thalamic and cortical areas of the brains of the two sheep used as bioassays for SSBP/1-challenged goat brain, but was absent or scarce in the medulla.
Detection of PrP by Western blotting
Despite the apparent absence of PrPSc in the SSBP/1 icc, PrPSc was detectable by Western blotting, using the 1B3 antibody, of brain from seven of the eight SSBP/1-infected, and all eight of the BSE-infected goats (Fig. 2). The eighth SSBP/1-infected goat, which was negative for PrPSc by Western blotting, was also negative by icc. Proteinase K-treated SSBP/1 samples had less intense signals than equivalently treated BSE samples; however, this method is not designed to provide a direct quantification of the amount of PrP in each sample, it simply shows presence or absence.
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Western blotting has also been carried out on goat and sheep experimental BSE and scrapie brain samples using the three other antibodies described for icc (BG4, FH11, 1A8). With 1A8, PrPSc detection levels were similar to those revealed by 1B3; however, non-specific background staining levels were much higher with 1A8. BG4 was slightly less sensitive, especially on scrapie PrP, whilst FH11 detection of proteinase K-treated PrP was very poor.
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Discussion |
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Sheep studies have previously recorded similarities between CH1641 and BSE. Foremost is the targeting of PrP genotypes of sheep following experimental challenge: both TSE isolates cause disease, with the shortest incubation periods being in QQ171 sheep (Goldmann et al., 1994 ). The PrPSc glycoform patterns isolated from the brain of CH1641- and BSE-affected sheep (Hope et al., 1999
) are also similar, although the two TSE sources differ markedly in mouse transmission characteristics. Here we have shown a further difference between these two isolates of TSEs.
In contrast to the icc results from SSBP/1, Western blotting indicated that PrPSc could be extracted from the brains of goats challenged both with SSBP/1 and with BSE. Although a direct comparison of PrP quantities from these protein blots may be misleading, it is evident that the intensity of staining of PrPSc is less for SSBP/1 than for BSE. The juxtaposing of results between positive PrPSc detection by Western blotting from SSBP/1 brain and the negative or nearly negative results following icc may reflect relative concentrations of protein detectable by the two methods. During tissue preparation for Western blots, protein is concentrated from substantially greater quantities of brain than those examined in icc sections. Although Western blot samples were taken from the thalamic area, some adjacent tissue may also have been included and the PrPSc fraction of the tissue was then concentrated. It is clear that the tissue sections used for icc have much less volume and so would be expected to contain very much less total PrPSc.
It is possible that immunoreactive epitopes on the PrPSc molecule may have remained undetected with icc. This could have occurred either because of the specific nature of the two monoclonal antibodies (FH11 and BG4) and possible variations in cleavage of the N terminus of PrPSc between SSBP/1 and BSE, or because some PrPSc epitopes remained masked, perhaps as a result of PrPSc protein structural conformation. The icc results achieved with FH11 and BG4 for known natural scrapie cases and the experimental BSE cases were similar and highly repeatable for distribution and intensity of PrPSc immunostaining. In an attempt to deal with this more fully, two polyclonal antisera (1A8 and 1B3) were used on a proportion of affected goats. The epitopes recognized by both 1A8 and 1B3 are extensive through much of the C-terminal part of the PrP molecule (see Methods); however, they revealed almost identical immunostaining to those of FH11 and BG4, confirming the former observations.
Taken together these results can be explained by PrPSc in goats generated from scrapie sources SSBP/1 and CH1641 having different conformations from that accumulating in BSE in goats. An association between the variation in PrPSc conformation and different infecting sources of TSE was recently demonstrated by Safar et al. (1998) .
There are also differences in the pattern of protein banding, indicating differing levels of each of the three possible glycoforms of the PrPSc protein between SSBP/1 and BSE. SSBP/1 appeared to generate appreciably more of the unglycosylated form of PrPSc and less of the diglycosylated form than is apparent with BSE, even for preparations of non-proteinase K-treated PrPSc. Whether these differences in glycosylation patterns between SSBP/1 and BSE could have been responsible for the dissimilarities in PrPSc immunorecognition observed with icc is unknown.
The glycoform pattern has been suggested as a possible means of distinguishing sources of TSE (Collinge et al., 1996 ; Hope et al., 1999
) and, although the protocol used for glycoform preparation here varied slightly to that used in the referenced studies, our results lend some support to that idea. However, it should be stated clearly that these differences in glycoform patterns between BSE and the experimental source SSBP/1 should not be taken as evidence of a test to distinguish between BSE and all natural scrapie. In fact a recent French study has shown that glycoform profiling of PrP from French natural scrapie cases produced very similar patterns to those from cattle with BSE and other experimental TSE cases in mice (Baron et al., 1999
).
Previous studies in sheep subcutaneously inoculated with SSBP/1 have produced weak immunostaining of PrPSc and minimal or no vacuolar degeneration in the brain of terminally affected animals (Foster et al., 1996 ). The present study has also shown quite clearly that PrPSc in TSE-affected goats can be at least partially dissociated from intense vacuolar degeneration, depending on the source of infection and means of PrP identification. Altogether, this indicates that in some instances of terminal TSE disease neither vacuolation nor PrPSc detection need necessarily be demonstrable.
The correlation between the appearance of PrPSc in infected tissue and TSE infectivity has been a matter of discussion for a long time. With the transmission of scrapie to sheep by inoculation of homogenized brain taken from the thalamic area of one of our SSBP/1-infected goats we have demonstrated the presence of infectivity in an area of intense vacuolation but little detectable PrPSc. Furthermore, the incubation periods of these bioassay sheep suggested no evidence of a significant reduction in infectivity titre compared with published data of SSBP/1 transmission in sheep (Goldmann et al., 1996 ), considering that both PrP genotype and the species was changed in these transmissions.
Our results, therefore, emphasize the importance of using several techniques for an unambiguous diagnosis of TSE infection. Reliance on a single method may lead to the misinterpretation of results by missing genuine TSE cases.
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Acknowledgments |
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References |
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Received 9 June 2000;
accepted 4 October 2000.