Institute for Animal Health, Compton, Newbury, Berkshire RG20 7NN, UK1
Neuropathogenesis Unit, Institute for Animal Health, Ogston Building, West Mains Road, Edinburgh EH9 3JF, UK2
Department of Zoology, South Parks Road, Oxford OX1 3PS, UK3
Author for correspondence: Matthew Baylis. Fax +44 1635 577237. e-mail matthew.baylis{at}bbsrc.ac.uk
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Abstract |
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Introduction |
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Three PrP polymorphisms have particularly strong linkage with the occurrence of both natural and experimental scrapie. These are valine (V) or alanine (A) at codon 136, arginine (R) or histidine (H) at codon 154, and glutamine (Q), arginine (R) or histidine (H) at codon 171 (reviewed by Hunter, 1997 ). Of the 12 possible alleles derivable from these polymorphisms, only five are commonly seen. These are: A136R154R171 (hereafter ARR), ARQ, VRQ, AHQ and ARH (Belt et al., 1995
). The ARR allele is clearly associated with resistance to scrapie and VRQ is clearly associated with susceptibility (Belt et al., 1995
; Hunter et al., 1996
). Sheep of VRQ/VRQ genotype are highly susceptible to the disease (Belt et al., 1995
; Hunter et al., 1996
) whereas sheep of ARR/ARR genotype appear to be resistant. Susceptibility of the ARQ/ARQ genotype is more complex and varies with sheep breed: in Suffolk sheep this is the most common genotype of scrapie cases (Hunter et al., 1997b
) while scrapie is rare in Texel sheep of this genotype (Dawson et al., 1998
). The AHQ allele may be associated with resistance in some breeds but not in others and the ARH allele may be neutral (Dawson et al., 1998
).
In August 1998 we took blood samples from all breeding animals in a pedigree flock of Texel sheep. At that time the farmer suspected that one sheep was showing signs of scrapie but had never previously reported a case to the relevant UK authorities. Over the next 17 months nearly 50 sheep were submitted to the authorities, and 20 cases of scrapie were confirmed. In December 1999 the farmer announced that the flock was to be dispersed, with the culling of susceptible individuals. Uniquely, therefore, we are able here to report the entire, official, epidemic of scrapie in this sheep flock and are able to answer several interesting questions. Was there evidence of the occurrence of scrapie in the flock prior to its first official reporting? What were the death rates from scrapie among the different susceptible PrP genotypes? And were particular PrP genotypes associated with any other (non-scrapie) fates?
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Methods |
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Tissue samples from suspect scrapie cases were subject to routine analysis for evidence of scrapie by the Veterinary Laboratories Agency, UK. Up to four methods were used: (i) histopathological examination of brain tissue for signs of vacuolation; detection of the disease-associated isoform of PrP (PrPSc) by (ii) immunocytochemistry (ICC) and (iii) Western blotting (WB); and (iv) detection of scrapie-associated fibrils (SAF).
For most analyses we consider only data collected between August 1998 and December 1999 (17 months). However, for survival analysis we include all available data, including deaths from scrapie between January and December 2000.
PrP genotype analysis.
For each sheep, approximately 5 ml of blood was collected into an EDTA-vacutainer and stored at -20 °C prior to genotype analysis. Genotype analysis was performed by DNA sequencing using an ABI Prism 377 DNA sequencer as recommended by the manufacturer. In short: DNA was isolated from 100500 µl blood using either a Nucleon DNA extraction kit (Anachem) followed by amplification/sequencing reactions as previously described in Baylis et al. (2000) , or a Qiagen DNeasy tissue extraction kit, after which approximately 1020% of the genomic DNA was subjected to 30 cycles of PCR amplification with oligonucleotide pair 218 CCGCTATCCACCTCAGGGA and 827 TTGCCCCTATCCTACTATGAGA. Following purification with Microcon columns (Amicon) PCR product was sequenced with oligonucleotides 4142 or 9612 (Baylis et al., 2000
) in Big Dye Terminator reagent (ABI) diluted in Better-Buffer (Microzone). Control samples of known genotypes were run in parallel in all PCR and sequencing reactions. Most scrapie samples were genotyped more than once. Results obtained in this manner represent pairs/groups of amino acids at three separate codon positions: 136 (valine, V, and alanine, A); 154 (histidine, H, and arginine, R); and 171 (glutamine, Q, arginine, R, and histidine, H). From this we infer that the PrP genotype, in allelic format is, for example, V136R154Q171/A136R154R171, because this is the only possible genotype derivable from the five known PrP alleles. All of the PrP genotyping performed at the Institute is consistent with the assumption that there are only five alleles with regard to these three codons in British sheep and so, for ease of interpretation, we have presented all genotypes in allelic format.
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Results |
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PrP genotypes of flock
Fifteen genotypes can be derived from the five known PrP alleles. Of these, 13 were found in the Texel flock (Table 1). About 70% of the flock were of just three genotypes: ARR/ARQ, ARQ/ARH and ARQ/ARQ. Only 15% of sheep carried the scrapie-associated VRQ allele. Significantly, there was only a single animal of the most highly susceptible VRQ/VRQ genotype. The genotype frequencies correspond to allele frequencies of: ARR, 22·7%; AHQ, 1·3%; ARQ, 50·9%; ARH, 17·4%; VRQ, 7·7%.
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Similar patterns may be obtainable by breeding susceptible (VRQ-encoding) ewes with resistant (ARR/ARR) rams. More convincing evidence of the prior occurrence of scrapie comes, therefore, from the age structure of the flock. The youngest sheep known to have died of scrapie in this flock were 2 years of age (next section). It is likely, then, that if there have been a substantial number of deaths from scrapie, there will be relatively fewer susceptible sheep over 3 years old than under 3 years old. This is the case (Table 2;
2=7·44, d.f.=4, P<0·025). Only 32% of sheep of the VRQ/ARQ, VRQ/ARH and VRQ/VRQ genotypes were between 3 and 8 years of age, compared to 61% for the other genotypes.
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The confirmation of scrapie in a sheep of genotype ARR/ARH (#12) is highly unusual. The genotype is consistent with its parentage (its sire was ARR/ARQ and its dam had at least one ARH allele) and is likely to be correct. It is possible that the confirmation of scrapie is, in fact, incorrect. The confirmation was based on a positive result by SAF, despite inconclusive histopathology and a negative result by ICC (Table 3). Confirmations of scrapie on the basis of SAF have been made in other instances where, subsequently, it has been shown that scrapie was very unlikely (M. Baylis, unpublished observations). The sensitivity of SAF as a diagnostic method for scrapie has been shown to be less than certain other methods (Simmons et al., 2000
) and, during 1999, VLA stopped confirmations of scrapie on the basis of a positive result by SAF alone. ICC and WB were introduced as alternatives (Table 3
). Independent Western blotting of tissue from this animal at the Neuropathogenesis Unit of IAH failed to detect PrPSc in either medulla or spleen. It seems likely that the confirmation of scrapie in animal #12 is a misdiagnosis.
Scrapie was confirmed in two other sheep on the basis of SAF only. Animal #6 was SAF-positive but negative by histopathology; it was ARQ/ARQ, a genotype in which scrapie occurs (albeit rarely) in this flock. This scrapie confirmation must be considered questionable. Animal #11 was SAF-positive but histopathology was inconclusive; it was VRQ/ARR, again a genotype in which scrapie occurs (rarely) in this flock. Independent Western blotting of tissue from the medulla of animal #11 at the Neuropathogenesis Unit of IAH detected PrPSc and the confirmation is considered valid.
Scrapie was confirmed in a second sheep of VRQ/ARR genotype (#14), on the basis of the detection of PrPSc by immunocytochemistry, despite negative results by histopathology and Western blotting. Independent Western blotting of tissue from the medulla of animal #14 at the Neuropathogenesis Unit of IAH also failed to detect PrPSc. The true scrapie status of this animal must be considered ambiguous. For the purposes here we consider the animal to have died of scrapie, but accept that this may be incorrect. Our conclusions are not significantly affected under either scenario.
Given the ambiguity in the true scrapie status of some of the confirmed cases, Table 3 indicates which cases we conclude to be true scrapie and include in further analyses. Of the original 20 confirmed cases (#120), 18 are considered to be scrapie although one (#6) is ambiguous. Two cases (#12, #13) are excluded from analyses.
Characteristics of the scrapie cases, grouped by genotype, are given in Table 4. The lowest median age of death, at 2·5 to 3 years, was for the VRQ/ARH and VRQ/ARQ genotypes; by contrast, the median age of death for the VRQ/ARR and ARQ/ARQ genotypes was nearly 4 years. Nevertheless, patterns relating genotype and age at death were not clear (for example, the range in age is almost identical for VRQ/ARH and ARQ/ARQ) and, overall, there was not a significant effect of genotype on the age at death (Mood median test,
2=3·9, df=3, not significant). This reflects, presumably, the range in age at which the sheep were first infected or the dose of infectivity received.
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Mortality and survival rates
In the 17 month period from August 1998 to December 1999, death rates from scrapie were: ARQ/ARQ, 3% (2/58); VRQ/ARR, 13% (2/16); VRQ/ARQ, 55% (6/11) and VRQ/ARH, 86% (6/7). Note that these rates exclude the two ram lambs that died of scrapie (#15, #16) and that were not included in the initial 233 sheep sampled in August 1998. Survival times of the sheep of these genotypes were subjected to survival analysis (Collett, 1994 ) using SAS (Allison, 1995
). The survival measure was time (in months) from August 1998 to the date of submission as a scrapie suspect (which was then confirmed at later date). KaplanMeier survival distribution functions for the four genotypes are shown in Fig. 1(A)
. There was a highly significant effect of genotype on survival function (Log rank test,
2=56·4, d.f.=3, P<0·0001), with survivorships of VRQ/ARQ and VRQ/ARH decreasing more rapidly than those of ARQ/ARQ and VRQ/ARR. Within these pairs, however, there were no significant differences in survivor function. It is noteworthy that the VRQ/ARQ and VRQ/ARH survivorships decrease to or close to zero, suggesting that the censored animals of those genotypes probably would have died from scrapie if they had not been lost from other causes.
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The genotypes ARH/ARH and ARQ/ARH have elsewhere been categorized as having similar risks of scrapie as ARQ/ARQ and VRQ/ARR (Dawson et al., 1998 ). It is perhaps noteworthy that there were four scrapie cases out of 74 sheep encoding ARQ/ARQ and VRQ/ARR, but none out of 54 sheep encoding ARH/ARH and ARQ/ARH. The difference approaches, but does not reach, significance (
2=3·0, d.f.=1, P<0·1).
Age-dependent survival
The oldest sheep of scrapie-susceptible genotype sampled in August 1998 was an ARQ/ARQ and almost 90 months old. Susceptible sheep were therefore divided into three groups according to their age in August 1998: 630 months old (note that we did not sample any sheep under 6 months old); 3060 months old; and 6090 months old. These age groupings were chosen arbitrarily. Because of small sample sizes, sheep of ARQ/ARQ and VRQ/ARR genotypes and VRQ/ARQ and VRQ/ARH genotypes were combined to give two groups of differing susceptibility to scrapie (low and high, respectively). We then used survival analysis to examine whether different cohorts had different survivorships (Fig. 1B). There were no deaths from scrapie in the youngest age cohort of the less susceptible sheep (thin solid line). Note that this analysis excludes the young ARQ/ARQ male that died of scrapie (#16) as it was not one of the 233 sheep sampled at the start of the study. Similarly, there were no losses from scrapie in the youngest age cohort of the more susceptible sheep (thick solid line) for the first 13 months. This was followed over the ensuing 10 months, however, by heavy losses from scrapie until there were no survivors. The middle and oldest age cohorts of the less susceptible sheep experienced some losses from scrapie in the early months of the study but then there were no others (thin dashed and dotted lines). The same age cohorts of the more susceptible sheep also experienced most losses in the early months of the study (thick dashed and dotted lines). Significantly, there appear to be no differences in the survival functions of the middle- and older-age cohorts in either group of sheep. In other words, the different ages of the cohorts and, possibly, their different durations of exposure to scrapie infectivity (depending on when the flock first became infected) do not appear to have affected their relative survivorships.
Unconfirmed suspect cases
During the 17 month study period, 30 animals were slaughtered and submitted to the authorities as scrapie suspects, but evidence of scrapie was not detected. One further negative case occurred after the decision to disperse the flock.
All of these cases showed some signs that occur with scrapie, most notably wasting. It is possible that at least some of these sheep may have had scrapie and were showing behavioural or physiological signs, but for unknown reasons confirmation in the laboratory was not achieved. If this occurred to any significant degree, we would expect there to be an association between failure to confirm scrapie and PrP genotype. This is not the case. Taking the survival measure to be time (in months) from August 1998 to the date of submission as a scrapie suspect (which was later not confirmed), there was no significant difference between the survival functions of sheep of the different scrapie risk categories given in Table 2 (Log rank test,
2=0·11, d.f.=2, P>0·5).
Deaths from other and unknown causes
Scrapie-affected farms report having more sheep that die of unknown causes than scrapie-free farms (McLean et al., 1999 ) and a high proportion of sheep that die from unknown causes may, in fact, show signs of scrapie when examined using histopathology (Clark et al., 1994
). It is interesting to ask, therefore, whether in our study farm there was an association between PrP genotype and the risk of being found dead.
During the 17 month study period, 18 sheep were found dead from unknown causes. Taking the survival measure to be time (in months) from August 1998 to the date of being found dead, there was no significant difference between the survival functions of sheep of the different scrapie risk categories given in Table 2 (Log rank test,
2=1·22, d.f.=2, P>0·5).
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Discussion |
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In responses to a questionnaire the farmer implied that there had been cases of scrapie in the flock prior to the start of our study. We have previously shown that scrapie leaves a statistically detectable signature in the age-genotype structure of scrapie-affected sheep flocks (Baylis et al., 2000 ). Here we confirm the presence of a signature in the study flock; namely, there was a significant paucity of older sheep of the most susceptible genotypes. One implication is that we were (at least in theory) able to conclude the likely occurrence of scrapie in the flock, even had information from the farmer not been made available.
A second signature was apparent in the observed and predicted frequencies of sheep of the VRQ/ARR genotype. The high level of susceptibility of VRQ-containing genotypes other than VRQ/ARR means that, as deaths from scrapie occurred in the flock, the frequency of the VRQ allele was reduced to a level too low to account for the observed number of surviving sheep of VRQ/ARR genotype. It remains to be seen whether this measure is a useful marker for the occurrence of VRQ-attacking scrapie in other sheep flocks.
If the flock had not been dispersed, it is likely that a high proportion of future scrapie cases would have been produced by either sires or dams of VRQ/ARR genotype, as sheep of the other VRQ-containing genotypes would be expected to die before reproducing many times. It is worth considering, therefore, that the high level of resistance of the VRQ/ARR genotype might have significantly extended the duration of the scrapie epidemic in the flock. This raises the intriguing possibility that the use of ARR/ARR (resistant) rams, with no consideration of ewe genotype, is not necessarily the most rapid method of eliminating scrapie from affected flocks.
There has been one previous study of scrapie in Texel sheep. Genotype frequencies were obtained by Belt et al. (1995) for scrapie-affected and unaffected Texel sheep collected from over 30 different flocks in The Netherlands. Scrapie was recorded in five different genotypes. It has been suggested by one of us previously that this large range in tropism is a result of the multi-flock nature of the study, because of variable host genetics or more than one scrapie strain, and that a simpler picture would be obtained in single flock studies (Hunter, 1997
). Our new data disagree with this assertion. In our single flock study, scrapie was recorded in four genotypes, and would be expected in a fifth (VRQ/VRQ). It is more likely, therefore, that the wide tropisms reported by Belt et al. (1995)
and here are characteristics of Texel sheep.
There are further interesting similarities between the results of the two studies. Belt et al. (1995) recorded four cases of scrapie out of 18 animals of ARQ/ARQ and VRQ/ARR genotype, but none in 20 animals of ARQ/ARH or ARH/ARH genotype. These proportions differ significantly (
2=5·0, d.f.=1, P<0·05). We observed the same pattern, although the results were not quite significant (0·05<P<0·1). These observations suggest that sheep of ARQ/ARH or ARH/ARH genotypes may be largely or entirely resistant to scrapie. This is in disagreement with the classification of these genotypes by Dawson et al. (1998)
and the UK Governments National Scrapie Plan (DEFRA, 2001
). The observed pattern might occur if, in Texel sheep, VRQ and ARQ are associated with scrapie and ARH is associated with resistance, and VRQ is dominant to both ARH and ARQ, while ARH is dominant to ARQ. Hence, scrapie would occur in VRQ/ARQ, VRQ/ARH and ARQ/ARQ genotypes, but not ARQ/ARH or ARH/ARH genotypes.
The wide scrapie tropism observed in Texel sheep differs significantly from that reported in other breeds. Scrapie has not been reported in the ARQ/ARQ and VRQ/ARR genotypes in the NPU flock of Cheviot sheep (Hunter et al., 1996 ). In a flock of Romanov sheep in France there were no confirmed cases in VRQ/ARR sheep but there were many in ARQ/ARQ sheep and the scrapie hazard for ARQ/ARQ was only one-third that of VRQ/VRQ and half that of VRQ/ARQ (Elsen et al., 1999
). By contrast, in our Texel flock scrapie was confirmed in both ARQ/ARQ and VRQ/ARR genotypes, but the hazard was about one-twentieth that of VRQ/ARQ.
There is experimental evidence for different scrapie incubation periods in different genotypes of the same breed of sheep (Goldmann et al., 1994 ), and this assumption has been a requirement for successful modelling of a within-flock scrapie epidemic (Matthews et al., 2001
). Generally, the shortest incubation periods are associated with the most susceptible genotypes. In the field, this pattern should be manifested as a negative relationship between susceptibility and average age at death, and this was observed in the epidemic in Romanov sheep (Elsen et al., 1999
). A similar pattern is apparent in our Texel sheep (Table 4
). The median age at death is lower for VRQ/ARQ and VRQ/ARH than ARQ/ARQ and VRQ/ARR cases. The difference was not significant, however, and this is because of the death of a single, young ARQ/ARQ male at 28 months (#16) and the deaths of two, old VRQ/ARH ewes at 77 and 82 months (#3, #8). The latter may simply be the result of infections acquired by the adult sheep. However, there can be no doubt that the case in the young ARQ/ARQ male suggests that incubation periods in this genotype of Texel sheep can be short, even though the level of susceptibility is low. An alternative explanation is that this case was caused by a second scrapie strain with different attack characteristics from the main strain attacking the flock.
There are several reasons for expecting the survival function of older sheep in a scrapie-affected flock to differ from that of younger sheep. First, younger sheep may be more susceptible to infection than older sheep (Matthews et al., 2001 ). Secondly, older and younger cohorts will be born at different stages in the epidemic and will probably be exposed, as lambs, to different levels of infectious agent. Higher infectious doses are generally associated with shorter incubation periods. With this in mind, it seems remarkable that in our Texel sheep the survival functions for middle and older age cohorts were almost identical, suggesting similar levels of susceptibility and time-courses to death.
Several studies have suggested that, in scrapie-affected sheep flocks, a number of the sheep found dead of unknown causes may have had undiagnosed scrapie. Were this the case, an association between the likelihood of being found dead of unknown causes and PrP genotype would be expected. In the current study there was a remarkable number of such sheep during the 17 month study period (8% of the entire flock), but there was not a significant association with scrapie risk.
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Acknowledgments |
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References |
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Received 26 March 2002;
accepted 9 July 2002.