1 Centre for Tropical Veterinary Medicine (CTVM), Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Roslin, Midlothian EH25 9RG, UK
2 Institute of Cell, Animal and Population Biology, University of Edinburgh, Edinburgh EH9 3JT, UK
3 Institut National de la Recherche Agronomique, Station d'Amélioration Génétique des Animaux, BP 27, 31326 Castanet-Tolosan Cedex, France
4 Institut National de la Recherche Agronomique, Unité de Mathématiques et Informatique Appliquées, 78352 Jouy-en-Josas Cedex, France
5 Sustainable Livestock Systems Group, Scottish Agricultural College, Bush Estate, Penicuik, Midlothan EH26 0PH, UK
6 Institute for Animal Health, Neuropathogenesis Unit, Ogston Building, West Mains Road, Edinburgh EH9 3JF, UK
Correspondence
Margo E. Chase-Topping
margo.chase{at}ed.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Despite recent detailed studies of scrapie outbreaks within individual sheep flocks (Elsen et al., 1999; Hunter et al., 1996
, 1997
) and comparative epidemiological analysis on multiple sheep flocks (Redman et al., 2002
), key determinants of epidemiological and transmission dynamics of sheep scrapie are still poorly understood. In recent years considerable progress has been made in establishing the genetics of susceptibility of scrapie (Dawson et al., 1998
; Hunter et al., 1997
). It is known that resistance or susceptibility is largely under genetic control (Hunter, 1997
), however, the effects of PrP genotype on scrapie susceptibility can vary between flocks and breeds of sheep (Dawson et al., 1998
) and can also depend on scrapie isolates (Goldmann et al., 1994
). To date, there have been few detailed within-flock studies of the effects of variation in PrP genotype at the individual level during natural scrapie outbreaks. Many studies have been performed to determine the genetic status and variability of PrP genotype of sheep breeds in different countries (e.g. Germany: Drogemuller et al., 2001
; Italy: Vaccari et al., 2001
and Spain: Acín et al., 2004
), and a few studies have examined the PrP genotype profile of individual flocks (e.g. Baylis et al., 2000
). Previous research that has examined genotype-level associations within flocks have generally focused on the relationship with scrapie infection, including incubation time (Goldmann et al., 1991
, 1994
) and age of onset of scrapie (Baylis et al., 2002
; Bossers et al., 1996
; Clouscard et al., 1995
; Elsen et al., 1999
). Despite the extensive amount of research that exists on scrapie infection no study has attempted to quantify the effect of scrapie on significant performance parameters such as lifetime breeding success (LBS), litter size or survival. A few analyses have examined PrP genotype-level associations with performance parameters (e.g. Brandsma et al., 2004
: litter size and 135 days weight; Barillet et al., 2002
: dairy production traits, Prokopova et al., 2002
: lean growth rate; and de Vries et al., 2004
: muscle mass, live-weight gain, wool quality and fat depth). Overall these studies have found no significant association between PrP genotype and the trait examined, although some association between the resistant ARR and depth of muscle mass was found in German black-headed mutton sheep (de Vries et al., 2004
). However, these studies examined the traits in the absence of scrapie infection, with a view to determining the effect of breeding for resistance, rather than the population dynamic and population genetic implications of a natural scrapie outbreak within a flock.
As with all TSEs, scrapie has a long incubation period between infection and onset of typical clinical signs. Although there is no explicit evidence to date for effects of pre-clinical scrapie, it has been identified as a possible cause for unexplained mortality within flocks (McLean et al., 1999). Furthermore, the focus of research on outbreaks of scrapie in sheep flocks has been on scrapie cases, and no study has considered individuals that did not develop clinical signs. Genotype-related differences in the performance of sheep manifesting no signs of scrapie may indicate the presence of pre-clinical scrapie within the flock. Identification and quantification of this phenomenon may result in changes in the incidence of scrapie deaths and the overall impact of scrapie as a disease within sheep.
In this paper, we focus on differences in individual performance associated with scrapie infection or PrP genotype in five different sheep flocks with natural outbreaks of scrapie. An outbreak of scrapie should exert substantial selection pressures against those PrP alleles associated with susceptibility. We illustrate the force of this selection by quantifying the effect of scrapie on individual fitness, assessed through estimates of individual LBS. Differences in LBS due to scrapie are expected within each flock. Such differences may be the result of differential longevity and/or differential fecundity. We examine each component across five different sheep flocks. Measures of individual LBS, litter size and survival are used to quantify: (i) the impact of scrapie; and (ii) differences between PrP genotypes in scrapie and non-scrapie-infected sheep.
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METHODS |
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For three of the outbreaks, the SAC Suffolk, the NPU Cheviot II and the INRA Romanov; there was some information on PrP genotypes, established by sequencing PCR products or by using oligonucleotide probes, as previously described (Elsen et al., 1999; Hunter et al., 1996
, 1997
). Data from these three flocks were used to examine genotype variation in LBS, litter size and survival. For the INRA Romanov flock, genotype data were available for all animals in the flock since the onset of scrapie in 1993, whereas the genotyped individuals in the SAC Suffolk and the NPU Cheviot II consisted mostly of scrapie cases and approximately 50 % of the non-scrapie-infected sheep in each flock. As such, the focus of the genotype variation analysis was on the INRA Romanov flock. However, where possible, corresponding data were presented for the NPU Cheviot II and SAC Suffolk flocks.
Statistical analysis.
Data within each database were standardized to suit the analysis that was to be performed. For all flocks experimental, non-breeding animals and all males were excluded from the analysis. In the INRA Romanov flock the breeding practices with males were different: replacement sires were not used for long, and experimental animals were mostly males and culled according to protocol. Males were therefore removed from the other flocks to standardize the data. Statistical analysis was first performed on each flock to determine differences associated with scrapie status (scrapie-infected versus non-scrapie-infected). For flocks with genotype information (INRA Romanov, NPU Cheviot II and SAC Suffolk), individuals were categorized as either susceptible (genotypes that were affected by scrapie) or non-affected (genotypes that were not affected by scrapie or scrapie infection was low or suspect). Susceptible and non-affected genotypes within each flock are listed in Table 2.
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Mean (±SE) LBS was estimated for each flock. Differences in the LBS of scrapie-infected and non-scrapie-infected sheep within each flock were analysed using a Student's t-test. To determine if there were any differences in the effect of scrapie on LBS across the five flocks, a comparison was performed using a Generalized Linear model (GLM) with negative binomial errors (S-Plus version 6.0) and the significance of the flock by status interaction was assessed from the change in deviance on dropping that term from the model, distributed as 2(4 d.f.). For flocks with genotype information, two analyses were performed to examine differences in LBS; the first examined differences between scrapie-infected and non-scrapie-infected individuals within and across susceptible genotypes and the second examined the LBS of non-scrapie-infected individuals, looking for differences between susceptible and non-affected genotypes. Both analyses were performed using a GLM (SAS version 8.2).
Variation in litter size.
The size of all litters produced throughout the scrapie outbreak was calculated for scrapie and non-scrapie dams in each flock. Data from all flocks were standardized to include all litters born within the years of the scrapie outbreak (Table 1).
Differences between scrapie and non-scrapie individuals and between PrP genotypes in the number of live lambs per litter (litter size) produced by dams at each breeding event were tested. Linear mixed effect models with dam identity fitted as a random effect were used to account for the repeated measures made on individual sheep over multiple breeding attempts. PrP genotype or scrapie status was used as a fixed effect. Models were fitted with Poisson errors using the procedure glmmPQL (S-Plus version 6). For all flocks, we initially tested for effects of breeding year (as a multilevel factor) and dam age (as a quadratic function). These variables, if significant (P<0·05), were retained in the models as follows: NPU Cheviot II, dam age; SAC Suffolks, breeding year; INRA Romanovs, NPU Cheviot I and NPU Suffolks, dam age and breeding year. Analyses of associations between litter size and PrP genotype were restricted to the INRA Romanov and SAC Suffolk flocks due to insufficient genotype data in the other flocks.
Variation in survival.
Survival analyses were performed on the female population considering the age at removal from flock as the survival measurement. Removal includes animals that died naturally as well as those culled for non-experimental reasons. Data analysed included only cohorts that were exposed to scrapie (Table 1). All survival analyses were performed using Proc Lifetest and Proc Phreg (SAS version 8.2). Median life expectancies (±95 % confidence intervals) were calculated using survival data censored for sheep culled at less than 1 year of age and those still alive. Data were stratified by genotype (VRQ/VRQ, ARQ/VRQ, ARQ/ARQ or non-affected) and scrapie status (scrapie or non-scrapie-infected). The following null hypotheses were tested in Proc Lifetest: (i) there are no differences in the overall mean life expectancy of scrapie-infected versus non-scrapie-infected individuals within each of the five flocks; and (ii) there are no differences in the mean life expectancy of non-scrapie-infected individuals among the susceptible and non-affected genotypes in the NPU Cheviot II, SAC Suffolk and INRA Romanov flocks. Differences between survivorship curves were tested using Kaplan-Meier estimator and the log-rank test. Significance was set at P
0·05, and where multiple comparisons were performed the Bonferroni correction was applied.
In addition to the Kaplan-Meier procedure, Cox proportional hazard models were run using Proc Phreg (SAS version 8.2) to determine the significance of any variables other than genotype in the survivorship of non-scrapie-infected individuals. Selection of variables was made by looking for significant changes in the log likelihood (2) after using a hierarchical method of variable selection (Collett, 2003
). The following variables were tested for significance and model improvement: year of birth, mode of feeding (maternal versus artificial) and breeding status (breeder or non-breeder). Genotype was added into the model last after other significant variables were adjusted for. Significance was set at P
0·05. Bestfit of all models was examined by looking at the residuals.
Variation in cause of removal.
Managers of the INRA Romanov flock kept records on the reason for removal from the flock in addition to the date of removal. The data can be grouped into the following three categories: poor health (e.g. mastitis, arthritis, septicaemia, lungs, diarrhoea, toxaemia), accidental (e.g. drowning, fracture, wound) and management (e.g. culled for meat, sold, age-related culling). Such data may provide information to indicate whether or not there are any removals that may be attributed to pre-clinical scrapie. We hypothesize that effects of pre-clinical scrapie would result in sheep with the susceptible genotypes being removed significantly more for health-related causes than sheep with non-affected genotypes. To test this hypothesis we examined the causes of removal in the three most susceptible genotypes (ARQ/VRQ, VRQ/VRQ and ARQ/ARQ) as well as the non-affected genotypes. Comparisons of the number of removals of susceptible and non-affected genotypes within each removal category were made using a 2 test or Fisher's exact test (if n<5). Analysis of frequency data were carried out in StatXact (version 5.0). Statistical significance was set at P
0·05.
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RESULTS |
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Association with PrP genotype.
For the INRA Romanov flock, we compared LBS in scrapie and non-scrapie-infected sheep within each of the three susceptible genotypes (ARQ/ARQ, ARQ/VRQ and VRQ/VRQ) (Fig. 1). The INRA Romanov flock had genotype information on all the scrapie-infected sheep (n=202) and the majority (67 %; n=330/491) of non-scrapie-infected sheep. Amongst susceptible genotypes a GLM revealed significant effects of both status (scrapie-infected versus non-scrapie-infected: F1,360=50·36, P<0·001) and of genotype (ARQ/ARQ, ARQ/VRQ and VRQ/VRQ; F2,360 P=0·004) on LBS. There was no interaction between the two factors (P=0·709), indicating that the proportionate reduction in LBS due to scrapie did not differ between genotypes. As observed across the entire flock, the LBS of scrapie-infected sheep was significantly less than non-scrapie-infected sheep. Regardless of status, multiple comparison tests (with Bonferroni correction) revealed that the LBS of VRQ/VRQ was significantly lower than both ARQ/ARQ (P=0·003) and ARQ/VRQ (P=0·022) but there was no significant difference between ARQ/ARQ and ARQ/VRQ (P=0·815).
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The NPU Cheviot II flock had genotype information on all scrapie-infected sheep (n=10), however, very few (18 %; n=41/225) non-scrapie-infected sheep were genotyped. Despite the small sample size a GLM analysis of status (scrapie-infected versus non-scrapie-infected) and genotype (ARQ/VRQ versus VRQ/VRQ) was performed amongst susceptible genotypes. There was no interaction between the two factors (P=0·696) and no significant status (P=0·083) or genotype differences (P=0·057) although genotype tended towards significance, with the LBS of VRQ/VRQ sheep less than that of sheep with the ARQ/VRQ genotype. Considering only non-scrapie-infected sheep, comparison of the LBS of the three susceptible and non-affected genotypes revealed significant genotype effects (P=0·002). Multiple comparisons (with Bonferroni correction) revealed that the LBS of non-scrapie-infected VRQ/VRQ sheep was significantly less than the non-affected genotypes (P=0·0036).
Within the SAC Suffolk flock there was only one susceptible genotype (ARQ/ARQ). As with the NPU Cheviot II flock, genotyping information was limited. All scrapie-infected sheep were genotyped however, only 39 % (n=211/537) of the non-scrapie-infected sheep were genotyped. Despite the small sample size, a one-way ANOVA on differences in the LBS of scrapie-infected versus non-scrapie-infected amongst ARQ/ARQ genotypes revealed no significant difference between scrapie-infected and non-scrapie-infected sheep within the susceptible genotype ARQ/ARQ (P=0·563). Considering only non-scrapie-infected sheep, there were significant differences between susceptible (ARQ/ARQ) and non-affected genotypes (P<0·001) where ARQ/ARQ sheep had a significantly lower LBS than the non-affected sheep.
Variation in litter size
Association with scrapie status.
The largest litter sizes were observed in the INRA Romanov flock and the smallest in the NPU Cheviot I flock (Table 4). There were no significant differences between the size of litters from scrapie-infected and non-scrapie-infected dams in each flock (Table 4
).
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Variation in survival
Association with scrapie status.
For all five flocks there was a significant reduction in the survival time (age at removal) of scrapie-infected individuals relative to non-scrapie-infected individuals (Table 5). The INRA Romanov had the largest difference between median survival of scrapie-infected and non-scrapie-infected sheep (4·3 years) whereas the NPU Cheviot I had the lowest (1·4 years).
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In the SAC Suffolk flock both the Kaplan-Meier (2=3·90, d.f.=1, P=0·048; Fig. 2c
) and Cox proportional hazards genotype-only model revealed significant differences among the two genotype groups (ARQ/ARQ and non-affected) in the age at removal of non-scrapie-infected sheep. The risk of early death for sheep with genotype ARQ/ARQ was 1·5 times higher than non-affected sheep but the significance was marginal (P=0·049). However, adjusting for significant variables (i) year of birth and (ii) breeding status revealed that differences between genotypes ARQ/ARQ and non-affected were significant (P=0·010).
Variation in cause of removal
For all three flocks examined there were genotype differences in the life expectancy of the sheep. Overall, sheep with highly susceptible genotypes did not live as long as sheep with non-affected and/or less susceptible genotypes. Examination of the distribution of age at death from scrapie (Fig. 2ac) revealed similarity between the three flocks. The peak in scrapie deaths approximates the point at which 50 % of the susceptible yet non-scrapie-infected animals in the flock are being removed (Fig. 2ac
). For example, in the INRA Romanov flock mean age of scrapie deaths is approximately 2 years of age, with all scrapie deaths occurring before age 4. In the survival graph for non-scrapie-infected deaths all VRQ/VRQ and ARQ/VRQ die within 4 years, whereas the less susceptible ARQ/ARQ and non-affected genotypes have a maximum lifespan of 9 years (Fig. 2a
). A similar pattern can be observed for VRQ/VRQ and ARQ/VRQ sheep in the NPU Cheviot II flock and the ARQ/ARQ in the SAC Suffolk flock.
For the INRA Romanov flock the presence of the VRQ allele appears to be a significant factor in the age at removal of non-scrapie-infected sheep in flocks affected by scrapie. The cause of this lower mean life expectancy in ARQ/VRQ and VRQ/VRQ sheep in the presence of scrapie suggests pre-clinical scrapie amongst the most susceptible genotypes. To explore this hypothesis further we examined the causes of death in non-scrapie-infected sheep with the highly susceptible genotypes (VRQ/VRQ and ARQ/VRQ) versus other non-scrapie-infected sheep with ARQ/ARQ and non-affected genotypes. A greater proportion of animals with the highly susceptible genotypes were removed for health-related reasons (2=41·11, d.f.=1, P<0·001), whereas animals with ARQ/ARQ and non-affected genotypes were more likely to be removed for management reasons (
2=38·56, d.f.=1, P<0·001). There was no significant difference between the genotype groups for the proportion of animals removed for accidental causes (P>0·05).
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DISCUSSION |
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There were significant differences in LBS of scrapie and non-scrapie-infected sheep within the four flocks where there was sufficient data to examine the comparison, with scrapie-infected sheep producing on average 34 % fewer offspring than non-scrapie-infected sheep. However, despite differences in the mean LBS measured in each flock, there was no evidence of any difference between flocks in proportionate reduction in LBS due to scrapie. There is therefore no indication of any variation between sheep breeds in loss of fitness due to scrapie infection. In addition to the overall effect of scrapie, there were also genotype differences in the LBS of scrapie and non-scrapie sheep, which correlated with the susceptibility of the genotype (VRQ/VRQ<ARQ/VRQ<ARQ/ARQ<non-affected). This could only be examined in detail for the INRA Romanov flock, but a similar pattern was apparent in the NPU Cheviot II and SAC Suffolk flocks.
The effect of scrapie on LBS appears to be a function of lifespan as opposed to fecundity. Analysis of litter size revealed no overall or genotype differences among the five sheep flocks. However, significant differences in survival of sheep were identified in this study. In general, age at removal from the flock depends on individual status (i.e. scrapie infection) and PrP genotype. For the five flocks examined, the median age at which scrapie-infected sheep were removed from the flock was significantly less than that for non-scrapie-infected sheep. Reduced survival in scrapie-infected sheep was expected based on previous research where lower life expectancies were observed for the most susceptible sheep in the flocks (Bossers et al., 1996; Clouscard et al., 1995
; Elsen et al., 1999
; Thorgeirsdottir et al., 2002
). As such, differences in the survival of scrapie-affected sheep was not analysed in detail in this study. The focus of the survival analysis in this study was on non-scrapie-infected sheep. The results of the survival analysis and Cox proportional hazard model indicated significant genotype differences in the pattern of survival among the non-scrapie-infected individuals for the flocks examined. Even when adjustment is made for significant covariates, there was an increased risk of removal associated with susceptible genotypes. For the INRA Romanov flock this seemed to depend on genotype or genotype susceptibility. VRQ/VRQ and ARQ/VRQ genotype individuals had significantly lower life expectancies, whereas the life expectancy of ARQ/ARQ genotyped sheep were not significantly different from non-affected sheep.
The distribution of removals from each flock approximates the age distribution of scrapie deaths. This distribution suggests that although scrapie was not diagnosed, these sheep were removed because of scrapie that was not detected or other health-related causes associated with scrapie incubating within the sheep. Reports from other field studies are inconsistent. McLean et al. (1999) reported having more sheep die of unknown causes on scrapie-affected farms than scrapie-free farms. Baylis et al. (2002)
also observed in scrapie-affected sheep flocks a number of sheep that were found dead of unknown causes (8 % of entire flock) but there was not a significant association with scrapie risk. In a recent study, however, a high prevalence of scrapie (6 %) was observed amongst sheep that were found dead in Shetland where scrapie is very common (Humphry et al., 2004
).
For the INRA Romanov flock a significantly higher proportion of ARQ/VRQ and VRQ/VRQ sheep died of poor health in comparison to ARQ/ARQ and the non-affected genotypes. One would have expected that if removals were the result of pre-clinical scrapie then sheep with the ARQ/ARQ genotype would also have a high proportion of removal as a result of health-related illness. It appears that there may be a deleterious effect of the presence of the VRQ allele in the presence of scrapie in the flock. Unfortunately there were no equivalent data from the other flocks with which to test this notion. The results of this study suggest that, across different flocks of different sheep breeds, susceptible PrP genotypes appear to perform less well in overall fecundity and life expectancy even if they do not contract scrapie. This effect is more apparent in the most susceptible genotype: VRQ/VRQ performed consistently worse in relation to LBS and survival even amongst apparently uninfected individuals.
There are two possible explanations for these findings. The first is that susceptible genotypes are in relatively poorer condition and are removed at younger ages. Unfortunately lack of data makes this hypothesis difficult to examine, although research to date suggests that there are no PrP genotype-related performance traits (Roden et al., 2001; Barillet et al., 2002
; Brandsma et al., 2004
; de Vries et al., 2004
). The second hypothesis is that they are suffering from effects of pre-clinical scrapie, which is manifesting itself in terms of reduced lifespan even though typical clinical signs of scrapie are yet to develop. If this hypothesis were true we might expect: (i) most deaths in years 24 when most scrapie cases occur; and (ii) the cause of death for susceptible genotypes to be different (i.e. more health-related). Both expectations are supported by the results reported here, although the results for the susceptible genotype ARQ/ARQ in the INRA Romanov flock are not as clear. Physiological evidence of pre-clinical scrapie does exist. Changes in behaviour that appear to consistently precede clinical signs have been observed (Parry, 1983
) and studies have shown that there was reduced rumination in sheep with scrapie and cattle with BSE (Austin & Simmons, 1993
). This reduced rumination may provide an explanation for the observations of loss of weight or body condition that has been reported for scrapie (Clark & Moar, 1992
), BSE (Wilesmith et al., 1992
) and chronic wasting disease (Williams & Young, 1982
).
Scrapie has become the target of control measures and eradication programmes worldwide. The identification of infected sheep is crucial for the success of these programmes. After initial infection, the disease has a long incubation period during which time infected sheep may be able to transmit disease to non-infected sheep. Evidence of scrapie can now be detected in sheep before the clinical signs occur (e.g. Schreuder et al., 1998) but it is unknown whether or not sheep are affected during this pre-clinical phase. This study has raised the possibility that reduced lifespan in susceptible PrP genotypes may be the result of pre-clinical scrapie. If pre-clinical scrapie does exist amongst susceptible genotypes we may underestimate levels of scrapie-related mortality in sheep flocks. The results presented here highlight the need for further research on performance of different sheep PrP genotypes both in the presence and absence of scrapie.
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ACKNOWLEDGEMENTS |
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Received 14 May 2004;
accepted 13 December 2004.
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