BACKGROUND
after the discovery that bovine spongiform encephalopathy  and variant creutzfeldt-jakob disease  were caused by the same agent  <cit>  research on transmissible spongiform encephalopathies  targeted other farm animal species that may harbour the bse agent.
sheep were a particular concern because scrapie, another tse, has been endemic in sheep flocks for more than  <dig> years, and if sheep had been fed bse infected meat and bone meal, there would have been the possibility that it could go undetected if mistaken for scrapie.
several studies have been conducted at the veterinary laboratories agency  since  <dig> to investigate the clinical presentation, pathology, pathogenesis, diagnosis and transmission of bse in sheep.
as for scrapie, the susceptibility of sheep to bse is largely determined by polymorphisms  of the gene that encodes the prion protein .
sheep most susceptible are homozygous for alanine at codon  <dig>  glutamine at codon  <dig>  and either arginine  or histidine  at codon  <dig>  <cit> , whereas arginine at codon  <dig>  is associated with increased resistance  <cit> .
although intracerebral infection of arr/arr sheep with bse has produced clinical disease  <cit> , it is not known whether oral infection can produce clinical disease in this genotype within the natural life span of sheep.
a more recent study proposed that other polymorphisms at codons  <dig>   <dig>   <dig> and  <dig> of the prp gene may also influence the susceptibility of sheep to experimental bse  <cit> .
despite considerable variation in the clinical signs of scrapie in sheep, three main types have been described, a pruritic form, a paralytic form without pruritic behaviour, and an atypical form, which usually includes signs of a cerebellar disease  <cit> .
it is largely unknown which factors determine the clinical presentation but it may be the result of differences between strains  <cit> , genotypes  <cit>  or clinical duration  <cit> .
other factors, such as breed, dose or age at infection may also be contributory; for example, differences in the vacuolar lesion profiles in scrapie have been attributed partly to the sheep breed  <cit> .
the first study conducted in the uk described bse in sheep as an acute disease with a duration of less than one week regardless of the route of inoculation , which was characterised by ataxia resulting in recumbency; pruritus was usually absent  <cit> .
this was subsequently confirmed in an oral transmission study  <cit> .
by contrast, intraperitoneal infection of a sheep with bse resulted in a protracted clinical course and at the late-stage intense pruritus and ataxia  <cit> , and in a dutch study four orally inoculated sheep developed clinical signs that included ataxia, head tremor and a nibble reflex  <cit> .
a more detailed clinical study of  <dig> sheep inoculated intracerebrally or intravenously with bse suggested that bse is "more likely to present with sudden onset of ataxia in the absence of pruritus compared to scrapie although 42% presented with pruritus alone and 25% displayed pruritus in combination with ataxia"  <cit> .
observed variations were attributed to the different routes of inoculation, different doses and different inocula .
the objective of this paper is to describe the clinical signs of bse in sheep, throughout its clinical course and at end-stage.
the bulk of the data concerns arq homozygous suffolk and romney sheep, which were orally challenged with the bse agent or acquired the infection naturally in an experimental flock.
a small number of other breeds and genotypes and two sheep inoculated intracerebrally were also studied.
to evaluate whether disease-associated clinical markers exist, the clinical features were compared with those of culled sheep of different genotypes and breeds that had not been challenged with bse  or had been challenged but not confirmed pathologically as bse cases.
for the purpose of this paper, a bse case is an animal that developed undisputable clinical features of bse and was positive on postmortem examination of the brain.
RESULTS
forty-seven sheep  were positive for bse by postmortem tests on brain tissue.
eleven of these were culled at pre-determined time points to study bse pathogenesis and tissue distribution.
the numbers of bse cases in sheep of each genotype and breed and the doses, routes of infection and incubation periods are detailed in table  <dig>  table  <dig> lists the details of unchallenged control sheep and challenged sheep without pathological confirmation of bse.
ip = incubation period in months , defined as time period from inoculation  to cull.
the mean incubation period with the standard error in parentheses is given if the numbers exceed four.
excluded are the scheduled culls  in the oral pathogenesis studies .
ip = incubation period in months , defined as time period from inoculation to cull and – for unchallenged animals in the oral bse pathogenesis study – from test group exposure and cull.
the mean incubation period with the standard error in parentheses is given if the numbers exceed four.
clinical assessment prior to cull
* p-values: + ≤  <dig> ;
++ ≤  <dig> ;
+++ ≤  <dig> ;
ns statistically not significant
the bse-negative subset included challenged and unchallenged animals.
the homogeneity of the clinical signs within this subset was proven by fisher's exact test
pruritus, defined as the occurrence of a positive scratch test  or – if this was not tested – a self-induced scratch response , occasional or frequent rubbing, scratching or nibbling , was present in  <dig>  out of  <dig> bse positive sheep.
in advanced cases in arq homozygotes, a clear nibbling response could sometimes be elicited by merely touching the sheep , whereas nibbling was less pronounced or required more forceful scratching in the ahq homozygous cheviots .
the clinical signs displayed by the six non-pruritic bse cases, which were culled at pre-determined time-points, are shown in table  <dig>
<dig> the display of a positive scratch test, a self-induced scratch response, self-nibbling, rubbing or scratching
hp = histopathological examination of the brain
ihc = immunohistochemical examination of the brain
neg./pos.
= negative/positive
n/a = not assessed
romney sheep were inoculated at approximately six months of age, suffolk sheep at approximately four months of age.
based on the criteria that a positive scratch test was an indication of bse infection in the brain , the test sensitivity was 84% and test specificity 98% .
of the six cases that were positive on postmortem tests without displaying a positive scratch test , three were culled at  <dig> months post inoculation  and displayed no specific signs  whilst the other three animals were killed between  <dig> and  <dig> mpi and displayed pruritic behaviour, such as frequent or occasional rubbing and nibbling in combination with alopecia in two and a self-induced nibble reflex in one animal.
one sheep with a positive scratch test on two consecutive examinations that had no detectable prpd in the brain and lymphoreticular tissue was a dosed arq/arq suffolk culled at  <dig> mpi, which did not display any other clinical signs except for one event of rubbing.
other pruritic behaviour  was observed in  <dig>  of  <dig> bse-negative sheep  that were observed sufficiently within four weeks prior to cull.
the genotype of these sheep was either arq/arr  or arr/arr ; all were dosed with the bse agent and culled between  <dig> and  <dig> mpi .
all confirmed bse cases that were ataxic prior to cull  also displayed pruritus, characterised by either a positive scratch test  or – if this was not tested  – by frequent rubbing, scratching and nibbling.
the combination of changed behaviour, pruritus and ataxia was displayed by  <dig>  of  <dig> bse positive cases that had each sign assessed but was not observed in bse-negative sheep.
over-reactivity to auditory stimuli, such as repeated flinching in response to a hand clap, was displayed by three  of  <dig> tested bse-affected sheep and none of the bse-negative sheep.
the assessment of cranial nerve function was unremarkable in most cases except for either unilateral or bilateral decreases in the menace response in three  of  <dig> bse-affected sheep .
sheep of all groups , particularly romney sheep, often exhibited a weak pupillary light response.
as visual deficits were not evident and no eye abnormalities were apparent by indirect ophthalmoscopy, this finding was considered to be normal.
intercurrent diseases
six sheep with at least one arr allele were culled with intercurrent diseases and had a negative bse test result on brain and lymphoreticular tissue.
a romney wether  presented with progressive weight loss, bruxism  during handling and alopecia on the bridge of the nose.
increased serum liver enzyme activity was suggestive of a liver disease; hepatitis was diagnosed histopathologically.
a suffolk wether  developed a head tremor at rest.
this sheep had a stiff, mildly ataxic gait and responded to scratching of the dorsum with lip tremor and head tilt .
haematology and blood biochemistry were unremarkable.
degenerative osteochondritis of the femoro-tibial joints and acute oedema of the brain suggestive of enterotoxaemia following clostridial replication was diagnosed, although there was no clinical and pathological evidence of enteritis.
another four sheep had displayed episodes of lateral recumbency with paddling of the limbs, which were described as "fits" by animal husbandry staff and may have been seizures.
a romney ewe  was found recumbent in the pen paddling with its limbs; the sheep had been unremarkable at a neurological examination a few days earlier.
an adenoma of the pituitary gland was diagnosed.
a romney ewe  had difficulty standing for  <dig> minutes after a sudden fit but had been clinically unremarkable before.
a leucoencephalopathy of undetermined but possible metabolic origin and an abscess at the base of the tongue were found.
a romney ewe  presented with weight loss, bruxism, head tremor and had a history of fits.
a diagnosis of leucoencephalopathy, possibly as a result of a metabolic disease, was made.
a romney wether  had a history of occasional fits since  <dig> mpi; these progressively became more frequent.
in addition, the sheep developed a left corneal oedema with ulceration but no other clinical signs were seen.
a leucoencephalopathy, similar to the previous two cases, was diagnosed.
clinical progression
* p-values: + ≤  <dig> ;
++ ≤  <dig> ;
+++ ≤  <dig> ;
ns statistically not significant
the analysis was compromised by the low number of cases, in particular those that had no detectable prpd in the brain, which displayed some of the clinical signs used for comparison, such as tremor or ataxia.
bse positive cases displayed most signs significantly earlier than bse-negative sheep, which included six undosed controls.
only the first display of fleece changes and bruxism during the examination when the animal was undisturbed  was not significantly different .
the clinical signs in  <dig> clinical suspects with confirmed bse that were monitored regularly from at least  <dig> weeks prior to cull are displayed in figures  <dig>   <dig>   <dig>   <dig>  signs usually progressed over a period of  <dig> weeks.
individual clinical signs were usually displayed in single animals only or expressed mildly  at  <dig> to  <dig> weeks prior to cull but became more frequent or severe as the disease progressed.
discussion
this study demonstrated that pruritus is a common feature in clinical cases of ovine bse.
forty-one  of the  <dig> bse-positive sheep displayed signs of pruritus.
a repeatable stereotypical response  to the scratch test, which has been associated with pruritus in sheep with scrapie  <cit> , was elicited in 85% of  <dig> bse-positive sheep.
the six non-pruritic bse cases, which were culled at pre-determined time points, displayed no specific signs of neurological disease; three had minor loss of weight prior to cull.
although the occurrence of even minor weight loss was significantly greater in clinical bse cases , this sign is not specific for bse and was also observed in bse-negative sheep at some stage during their life.
thus, we concluded that these six sheep represented pre-clinical bse cases.
all six sheep were culled between  <dig> and  <dig> mpi, and prpd accumulation was detected by immunohistochemical examination of parts of the central nervous system   <cit> ; three did not present with disease-specific vacuolation in the brain.
as most previous studies of experimental tse in sheep do not include detailed clinical assessments, it is difficult to know how frequently pre-clinical cases may be detected by postmortem brain tests.
in one study, orally dosed sheep with scrapie displayed early clinical signs around the same time as sparse prpd accumulation was detectable in the cns whereas one naturally infected sheep displayed no clinical signs and presented with vacuolation and prpd accumulation in the brain  <cit> , similarly to our three non-pruritic bse cases.
one sheep displayed a positive scratch test on two consecutive examinations prior to cull, which was highly suggestive of bse, even though prpd was not detected in the brain or lymphoreticular tissues of this sheep.
aside from tses, a positive scratch response may be elicited in animals with pruritic skin conditions, such as psoroptic mange  <cit> .
this sheep showed no significant pruritic behaviour, alopecia or skin lesions indicative of pruritus.
additionally, searches for ectoparasites remained negative in several "suspect" sheep tested at various time points throughout the studies, suggesting that our sheep remained ectoparasite free.
other pruritic behaviour  was also observed in  <dig> challenged sheep with at least one arr allele, which were not confirmed as bse cases by postmortem tests.
clinical and neurological signs have been reported in experimentally challenged arr/arr sheep with detectable prpd in the brain  <cit> .
if the pruritic behaviour observed in our sheep was indeed caused by bse we would have expected that the disease was confirmed by postmortem tests.
a clinical diagnosis of tse is more likely to be made if unequivocal neurological signs, such as head tremor or ataxia are present.
in our studies, several sheep developed skin lesions that led to culling for welfare reasons at a time point preceding the onset of ataxia in their cohort challenged group.
the severity of the skin lesions may not always reflect disease progression, given the influence of other factors, such as the presence of hard sharp surfaces in the pen and the sheep's individual propensity to rub or self mutilate.
in addition, clinical progression could not be monitored in the eleven bse cases that were culled at pre-determined time points to study bse pathogenesis and tissue distribution.
however, the clinical signs displayed by these sheep up to their cull date were identical to those noted in their cohort challenge group, contributing to the establishment of the clinical phenotype of bse in each challenge group.
six sheep inoculated with the bse agent were culled due to intercurrent diseases, five of which displayed neurological signs without pruritus.
all of these six sheep were either homozygous or heterozygous r at codon  <dig>  and postmortem tests showed no evidence of bse, as assessed by the absence prpd in the brain and lymphoreticular tissue of these sheep, and none of the sheep in our studies with this genotype have so far had bse confirmed by postmortem tests.
three sheep with histopathological evidence of leucoencephalopathy of undetermined origin presented with episodes resembling seizures although loss of consciousness, salivation, urination or defecation, which usually accompany seizures  <cit> , were not described.
seizures are most frequently associated with focal or diffuse forebrain or thalamic lesions caused by infections, toxins, tumours, nutritional deficiencies or metabolic diseases  <cit> .
a blood sample was not examined for the presence of metabolic disease  but this as well as toxins were considered unlikely because of the strict feeding and housing regime used in our studies.
our findings that sheep with bse are generally pruritic whereas ataxia is displayed later in the clinical course are in contrast to the observations of others  <cit> .
it is possible that the route of inoculation  results in a different clinical presentation compared to parenteral inoculation mostly used by others.
pruritus was not observed in a limited number of bse cases described in previous studies, but some of the sheep were kept at pasture and the methods by which clinical signs were assessed or recorded were not detailed  <cit> .
the very detailed clinical monitoring in our studies likely contributed to the detection of signs of pruritus, which could have been overlooked in another setting.
during our prolonged passive observations, we noted that the sheep would initially "freeze" and just stare at the observer, and that they would return to their "normal" group activities only after the first 10– <dig> minutes of observation.
this "freezing behaviour" explains why pruritus was seldom witnessed during the shorter daily or routine husbandry procedures, even in cases that had displayed pruritus for some time during passive observation sessions.
continuous video surveillance may be a good alternative to prolonged observations, since it has been shown that initial signs of pruritus were detected by video surveillance but were not apparent to observers  <cit> .
another useful contribution to the detection of signs of pruritus was the systematic assessment of the scratch test, which was not used in previously reported studies of bse in sheep.
one bse-affected sheep, which presented with a positive scratch test, tremor and changed behaviour , did not start pruritic activity until  <dig> minutes into an observation session, indicating that episodes of pruritic activity can be missed if sheep are observed for only a short time.
pruritic activity may be increased in association with resting behaviour in sheep.
disturbances caused by noise or the presence of humans result in a decrease in resting behaviour and an increase in movement and head alert reactions in sheep  <cit> , likely more so in nervous sheep.
when the presence of each individual clinical sign was compared between bse-positive and negative cases over the monitoring period from approximately  <dig> mpi until cull,  <dig> of  <dig> signs appeared significantly earlier  in bse positive cases.
for example, all positive cases with observed bruxism during handling displayed this sign before  <dig> weeks post inoculation  whilst approximately 89% of the bse-negative cases presented with this sign after  <dig> weeks.
only fleece changes and bruxism during the examination when the sheep was undisturbed  were not displayed earlier in positive or negative cases.
both are very unspecific signs and may occur in "normal" animals.
in addition, every statistical analysis is dependent on the number of observations.
a low number of observations, as seen for "bruxism when free", results in wide confidence intervals or prevents meaningful statistical analysis, which is why the degree of rubbing or nibbling was also not taken into consideration for the survival analysis, although frequent rubbing or nibbling was rarely or never observed in bse-negative cases  prior to cull.
loss of weight was one of the first observed signs of bse and was seen significantly earlier in bse-positive cases, although it was also present in inoculated animals that tested negative for bse and in some control sheep .
it frequently coincided with pruritic behaviour in bse cases.
thus, limiting the clinical assessment to weighing and a scratch test can be done fairly quickly and may be an efficient way to screen sheep for bse.
it was not investigated whether age at dosing affected the clinical presentation.
most of the sheep were challenged between four and six months of age, and the number of animals challenged at an older  or younger age  was too small to allow any statistical analysis.
a separate study conducted in the uk where sheep were challenged with bse at various ages has not found any obvious link between age of challenge, incubation period and type of clinical sign  <cit> .
the potential for discriminating tse strains by the clinical presentation was demonstrated in a mouse model where different prion strains produced different behavioural changes  <cit> .
the authors hypothesised that this may also apply to larger animals although the range of tests that can be performed on sheep and cattle is obviously limited by the size and cooperation of the animals.
bse in cattle  is a neurological disease with a fairly uniform clinical presentation characterised by changes in behaviour, sensation and locomotion  <cit>  and thought to be caused by a uniform strain based on a similarly uniform distribution and severity of vacuolar changes in cattle  <cit>  and mice  <cit> .
if strains predominantly dictate the clinical presentation, infection of sheep with the bse agent should equally result in a relatively uniform clinical disease, regardless of breed and genotype, since the pathological phenotype, including the intracellular truncation pattern, remains constant when bse is transmitted to sheep with different genotypes and by different routes  <cit> .
our results show that bse in sheep indeed presents with a relatively uniform clinical disease with pruritus as the predominant and initial sign, accompanied by behavioural and later locomotor changes as the disease progresses, and only rarely deficits of the menace response.
the two arq/arq suffolk sheep inoculated intracerebrally displayed pruritus in a similar fashion to the bse cases infected orally or naturally.
a temporal distribution in the display of clinical signs has also been shown in sheep scrapie, with an increased frequency of altered mental status, pruritus with wool loss, teeth grinding, head tremor, ataxia, loss of bodily condition and an absent menace response as the disease progresses  <cit> .
our findings suggest that the clinical presentation of bse in sheep of the susceptible or affected genotypes is largely influenced by the strain rather than breed, prp genotype or route of inoculation although the vast majority of bse cases were orally challenged arq/arq romney or suffolk sheep.
more cases in other breeds or genotypes including orally inoculated vrq/vrq sheep need to be studied.
natural infection, although only described in two cases in an experimental flock, did present with similar clinical signs and incubation periods.
no other classical scrapie strain has yet been found in sheep with similar uniform neuropathological, molecular and biological characteristics as bse.
clinical cases of atypical scrapie, which may be caused by a single strain based on the neuropathological and molecular characteristics  <cit> , display predominantly behavioural and locomotor abnormalities  <cit>  but only very limited cases have been studied so far.
well-characterised scrapie isolates, such as sspb/ <dig>  <cit> , produce a disease with variable clinical presentation in which pruritus and ataxia are displayed inconsistently in sheep of different breeds and genotypes inoculated subcutaneously  <cit> .
the variation seen in the neuroanatomical distribution of vacuolar changes and prpd accumulation in the brain of sheep with scrapie is believed to be affected not only by the scrapie strain but also by sheep breed and host genotype  <cit> .
differentiation of ovine bse from scrapie may not be possible based on clinical signs but our findings suggest that a scrapie suspect without signs of pruritus, such as the majority of atypical scrapie cases  <cit> , is unlikely to have bse.
CONCLUSIONS
oral bse infection of romney and suffolk arq/arq sheep causes a uniform disease with early pruritus followed by behavioural changes and movement disorders as the disease progresses.
identical findings in a limited number of additional bse cases described here suggest that the clinical features are independent of breed, genotype or route of inoculation.
it is hypothesised that the clinical presentation in sheep is predominantly influenced by the tse strain.
screening susceptible sheep for bse may be done efficiently by regular monitoring of their weights and testing them for a scratch response.

 after the discovery that bovine spongiform encephalopathy and variant creutzfeldt-jakob disease were caused by the same agent research on transmissible spongiform encephalopathies targeted other farm animal species that may harbour the bse agent. sheep were a particular concern because scrapie, another tse, has been endemic in sheep flocks for more than years, and if sheep had been fed bse infected meat and bone meal, there would have been the possibility that it could go undetected if mistaken for scrapie. several studies have been conducted at the veterinary laboratories agency since to investigate the clinical presentation, pathology, pathogenesis, diagnosis and transmission of bse in sheep. as for scrapie, the susceptibility of sheep to bse is largely determined by polymorphisms of the gene that encodes the prion protein. sheep most susceptible are homozygous for alanine at codon glutamine at codon and either arginine or histidine at codon, whereas arginine at codon is associated with increased resistance. although intracerebral infection of arr/arr sheep with bse has produced clinical disease, it is not known whether oral infection can produce clinical disease in this genotype within the natural life span of sheep. a more recent study proposed that other polymorphisms at codons and of the prp gene may also influence the susceptibility of sheep to experimental bse. despite considerable variation in the clinical signs of scrapie in sheep, three main types have been described, a pruritic form, a paralytic form without pruritic behaviour, and an atypical form, which usually includes signs of a cerebellar disease. it is largely unknown which factors determine the clinical presentation but it may be the result of differences between strains, genotypes or clinical duration. other factors, such as breed, dose or age at infection may also be contributory; for example, differences in the vacuolar lesion profiles in scrapie have been attributed partly to the sheep breed. the first study conducted in the uk described bse in sheep as an acute disease with a duration of less than one week regardless of the route of inoculation, which was characterised by ataxia resulting in recumbency; pruritus was usually absent. this was subsequently confirmed in an oral transmission study. by contrast, intraperitoneal infection of a sheep with bse resulted in a protracted clinical course and at the late-stage intense pruritus and ataxia, and in a dutch study four orally inoculated sheep developed clinical signs that included ataxia, head tremor and a nibble reflex. a more detailed clinical study of sheep inoculated intracerebrally or intravenously with bse suggested that bse is "more likely to present with sudden onset of ataxia in the absence of pruritus compared to scrapie although 42% presented with pruritus alone and 25% displayed pruritus in combination with ataxia". observed variations were attributed to the different routes of inoculation, different doses and different inocula. the objective of this paper is to describe the clinical signs of bse in sheep, throughout its clinical course and at end-stage. the bulk of the data concerns arq homozygous suffolk and romney sheep, which were orally challenged with the bse agent or acquired the infection naturally in an experimental flock. a small number of other breeds and genotypes and two sheep inoculated intracerebrally were also studied. to evaluate whether disease-associated clinical markers exist, the clinical features were compared with those of culled sheep of different genotypes and breeds that had not been challenged with bse or had been challenged but not confirmed pathologically as bse cases. for the purpose of this paper, a bse case is an animal that developed undisputable clinical features of bse and was positive on postmortem examination of the brain.  forty-seven sheep were positive for bse by postmortem tests on brain tissue. eleven of these were culled at pre-determined time points to study bse pathogenesis and tissue distribution. the numbers of bse cases in sheep of each genotype and breed and the doses, routes of infection and incubation periods are detailed in table table lists the details of unchallenged control sheep and challenged sheep without pathological confirmation of bse. ip = incubation period in months, defined as time period from inoculation to cull. the mean incubation period with the standard error in parentheses is given if the numbers exceed four. excluded are the scheduled culls in the oral pathogenesis studies. ip = incubation period in months, defined as time period from inoculation to cull and – for unchallenged animals in the oral bse pathogenesis study – from test group exposure and cull. the mean incubation period with the standard error in parentheses is given if the numbers exceed four. clinical assessment prior to cull * p-values: + ≤; ++ ≤; +++ ≤; ns statistically not significant the bse-negative subset included challenged and unchallenged animals. the homogeneity of the clinical signs within this subset was proven by fisher's exact test pruritus, defined as the occurrence of a positive scratch test or – if this was not tested – a self-induced scratch response, occasional or frequent rubbing, scratching or nibbling, was present in out of bse positive sheep. in advanced cases in arq homozygotes, a clear nibbling response could sometimes be elicited by merely touching the sheep, whereas nibbling was less pronounced or required more forceful scratching in the ahq homozygous cheviots. the clinical signs displayed by the six non-pruritic bse cases, which were culled at pre-determined time-points, are shown in table the display of a positive scratch test, a self-induced scratch response, self-nibbling, rubbing or scratching hp = histopathological examination of the brain ihc = immunohistochemical examination of the brain neg./pos. = negative/positive n/a = not assessed romney sheep were inoculated at approximately six months of age, suffolk sheep at approximately four months of age. based on the criteria that a positive scratch test was an indication of bse infection in the brain, the test sensitivity was 84% and test specificity 98%. of the six cases that were positive on postmortem tests without displaying a positive scratch test, three were culled at months post inoculation and displayed no specific signs whilst the other three animals were killed between and mpi and displayed pruritic behaviour, such as frequent or occasional rubbing and nibbling in combination with alopecia in two and a self-induced nibble reflex in one animal. one sheep with a positive scratch test on two consecutive examinations that had no detectable prpd in the brain and lymphoreticular tissue was a dosed arq/arq suffolk culled at mpi, which did not display any other clinical signs except for one event of rubbing. other pruritic behaviour was observed in of bse-negative sheep that were observed sufficiently within four weeks prior to cull. the genotype of these sheep was either arq/arr or arr/arr; all were dosed with the bse agent and culled between and mpi. all confirmed bse cases that were ataxic prior to cull also displayed pruritus, characterised by either a positive scratch test or – if this was not tested – by frequent rubbing, scratching and nibbling. the combination of changed behaviour, pruritus and ataxia was displayed by of bse positive cases that had each sign assessed but was not observed in bse-negative sheep. over-reactivity to auditory stimuli, such as repeated flinching in response to a hand clap, was displayed by three of tested bse-affected sheep and none of the bse-negative sheep. the assessment of cranial nerve function was unremarkable in most cases except for either unilateral or bilateral decreases in the menace response in three of bse-affected sheep. sheep of all groups, particularly romney sheep, often exhibited a weak pupillary light response. as visual deficits were not evident and no eye abnormalities were apparent by indirect ophthalmoscopy, this finding was considered to be normal. intercurrent diseases six sheep with at least one arr allele were culled with intercurrent diseases and had a negative bse test result on brain and lymphoreticular tissue. a romney wether presented with progressive weight loss, bruxism during handling and alopecia on the bridge of the nose. increased serum liver enzyme activity was suggestive of a liver disease; hepatitis was diagnosed histopathologically. a suffolk wether developed a head tremor at rest. this sheep had a stiff, mildly ataxic gait and responded to scratching of the dorsum with lip tremor and head tilt. haematology and blood biochemistry were unremarkable. degenerative osteochondritis of the femoro-tibial joints and acute oedema of the brain suggestive of enterotoxaemia following clostridial replication was diagnosed, although there was no clinical and pathological evidence of enteritis. another four sheep had displayed episodes of lateral recumbency with paddling of the limbs, which were described as "fits" by animal husbandry staff and may have been seizures. a romney ewe was found recumbent in the pen paddling with its limbs; the sheep had been unremarkable at a neurological examination a few days earlier. an adenoma of the pituitary gland was diagnosed. a romney ewe had difficulty standing for minutes after a sudden fit but had been clinically unremarkable before. a leucoencephalopathy of undetermined but possible metabolic origin and an abscess at the base of the tongue were found. a romney ewe presented with weight loss, bruxism, head tremor and had a history of fits. a diagnosis of leucoencephalopathy, possibly as a result of a metabolic disease, was made. a romney wether had a history of occasional fits since mpi; these progressively became more frequent. in addition, the sheep developed a left corneal oedema with ulceration but no other clinical signs were seen. a leucoencephalopathy, similar to the previous two cases, was diagnosed. clinical progression * p-values: + ≤; ++ ≤; +++ ≤; ns statistically not significant the analysis was compromised by the low number of cases, in particular those that had no detectable prpd in the brain, which displayed some of the clinical signs used for comparison, such as tremor or ataxia. bse positive cases displayed most signs significantly earlier than bse-negative sheep, which included six undosed controls. only the first display of fleece changes and bruxism during the examination when the animal was undisturbed was not significantly different. the clinical signs in clinical suspects with confirmed bse that were monitored regularly from at least weeks prior to cull are displayed in figures signs usually progressed over a period of weeks. individual clinical signs were usually displayed in single animals only or expressed mildly at to weeks prior to cull but became more frequent or severe as the disease progressed. discussion this study demonstrated that pruritus is a common feature in clinical cases of ovine bse. forty-one of the bse-positive sheep displayed signs of pruritus. a repeatable stereotypical response to the scratch test, which has been associated with pruritus in sheep with scrapie, was elicited in 85% of bse-positive sheep. the six non-pruritic bse cases, which were culled at pre-determined time points, displayed no specific signs of neurological disease; three had minor loss of weight prior to cull. although the occurrence of even minor weight loss was significantly greater in clinical bse cases, this sign is not specific for bse and was also observed in bse-negative sheep at some stage during their life. thus, we concluded that these six sheep represented pre-clinical bse cases. all six sheep were culled between and mpi, and prpd accumulation was detected by immunohistochemical examination of parts of the central nervous system; three did not present with disease-specific vacuolation in the brain. as most previous studies of experimental tse in sheep do not include detailed clinical assessments, it is difficult to know how frequently pre-clinical cases may be detected by postmortem brain tests. in one study, orally dosed sheep with scrapie displayed early clinical signs around the same time as sparse prpd accumulation was detectable in the cns whereas one naturally infected sheep displayed no clinical signs and presented with vacuolation and prpd accumulation in the brain, similarly to our three non-pruritic bse cases. one sheep displayed a positive scratch test on two consecutive examinations prior to cull, which was highly suggestive of bse, even though prpd was not detected in the brain or lymphoreticular tissues of this sheep. aside from tses, a positive scratch response may be elicited in animals with pruritic skin conditions, such as psoroptic mange. this sheep showed no significant pruritic behaviour, alopecia or skin lesions indicative of pruritus. additionally, searches for ectoparasites remained negative in several "suspect" sheep tested at various time points throughout the studies, suggesting that our sheep remained ectoparasite free. other pruritic behaviour was also observed in challenged sheep with at least one arr allele, which were not confirmed as bse cases by postmortem tests. clinical and neurological signs have been reported in experimentally challenged arr/arr sheep with detectable prpd in the brain. if the pruritic behaviour observed in our sheep was indeed caused by bse we would have expected that the disease was confirmed by postmortem tests. a clinical diagnosis of tse is more likely to be made if unequivocal neurological signs, such as head tremor or ataxia are present. in our studies, several sheep developed skin lesions that led to culling for welfare reasons at a time point preceding the onset of ataxia in their cohort challenged group. the severity of the skin lesions may not always reflect disease progression, given the influence of other factors, such as the presence of hard sharp surfaces in the pen and the sheep's individual propensity to rub or self mutilate. in addition, clinical progression could not be monitored in the eleven bse cases that were culled at pre-determined time points to study bse pathogenesis and tissue distribution. however, the clinical signs displayed by these sheep up to their cull date were identical to those noted in their cohort challenge group, contributing to the establishment of the clinical phenotype of bse in each challenge group. six sheep inoculated with the bse agent were culled due to intercurrent diseases, five of which displayed neurological signs without pruritus. all of these six sheep were either homozygous or heterozygous r at codon and postmortem tests showed no evidence of bse, as assessed by the absence prpd in the brain and lymphoreticular tissue of these sheep, and none of the sheep in our studies with this genotype have so far had bse confirmed by postmortem tests. three sheep with histopathological evidence of leucoencephalopathy of undetermined origin presented with episodes resembling seizures although loss of consciousness, salivation, urination or defecation, which usually accompany seizures, were not described. seizures are most frequently associated with focal or diffuse forebrain or thalamic lesions caused by infections, toxins, tumours, nutritional deficiencies or metabolic diseases. a blood sample was not examined for the presence of metabolic disease but this as well as toxins were considered unlikely because of the strict feeding and housing regime used in our studies. our findings that sheep with bse are generally pruritic whereas ataxia is displayed later in the clinical course are in contrast to the observations of others. it is possible that the route of inoculation results in a different clinical presentation compared to parenteral inoculation mostly used by others. pruritus was not observed in a limited number of bse cases described in previous studies, but some of the sheep were kept at pasture and the methods by which clinical signs were assessed or recorded were not detailed. the very detailed clinical monitoring in our studies likely contributed to the detection of signs of pruritus, which could have been overlooked in another setting. during our prolonged passive observations, we noted that the sheep would initially "freeze" and just stare at the observer, and that they would return to their "normal" group activities only after the first 10– minutes of observation. this "freezing behaviour" explains why pruritus was seldom witnessed during the shorter daily or routine husbandry procedures, even in cases that had displayed pruritus for some time during passive observation sessions. continuous video surveillance may be a good alternative to prolonged observations, since it has been shown that initial signs of pruritus were detected by video surveillance but were not apparent to observers. another useful contribution to the detection of signs of pruritus was the systematic assessment of the scratch test, which was not used in previously reported studies of bse in sheep. one bse-affected sheep, which presented with a positive scratch test, tremor and changed behaviour, did not start pruritic activity until minutes into an observation session, indicating that episodes of pruritic activity can be missed if sheep are observed for only a short time. pruritic activity may be increased in association with resting behaviour in sheep. disturbances caused by noise or the presence of humans result in a decrease in resting behaviour and an increase in movement and head alert reactions in sheep, likely more so in nervous sheep. when the presence of each individual clinical sign was compared between bse-positive and negative cases over the monitoring period from approximately mpi until cull, of signs appeared significantly earlier in bse positive cases. for example, all positive cases with observed bruxism during handling displayed this sign before weeks post inoculation whilst approximately 89% of the bse-negative cases presented with this sign after weeks. only fleece changes and bruxism during the examination when the sheep was undisturbed were not displayed earlier in positive or negative cases. both are very unspecific signs and may occur in "normal" animals. in addition, every statistical analysis is dependent on the number of observations. a low number of observations, as seen for "bruxism when free", results in wide confidence intervals or prevents meaningful statistical analysis, which is why the degree of rubbing or nibbling was also not taken into consideration for the survival analysis, although frequent rubbing or nibbling was rarely or never observed in bse-negative cases prior to cull. loss of weight was one of the first observed signs of bse and was seen significantly earlier in bse-positive cases, although it was also present in inoculated animals that tested negative for bse and in some control sheep. it frequently coincided with pruritic behaviour in bse cases. thus, limiting the clinical assessment to weighing and a scratch test can be done fairly quickly and may be an efficient way to screen sheep for bse. it was not investigated whether age at dosing affected the clinical presentation. most of the sheep were challenged between four and six months of age, and the number of animals challenged at an older or younger age was too small to allow any statistical analysis. a separate study conducted in the uk where sheep were challenged with bse at various ages has not found any obvious link between age of challenge, incubation period and type of clinical sign. the potential for discriminating tse strains by the clinical presentation was demonstrated in a mouse model where different prion strains produced different behavioural changes. the authors hypothesised that this may also apply to larger animals although the range of tests that can be performed on sheep and cattle is obviously limited by the size and cooperation of the animals. bse in cattle is a neurological disease with a fairly uniform clinical presentation characterised by changes in behaviour, sensation and locomotion and thought to be caused by a uniform strain based on a similarly uniform distribution and severity of vacuolar changes in cattle and mice. if strains predominantly dictate the clinical presentation, infection of sheep with the bse agent should equally result in a relatively uniform clinical disease, regardless of breed and genotype, since the pathological phenotype, including the intracellular truncation pattern, remains constant when bse is transmitted to sheep with different genotypes and by different routes. our results show that bse in sheep indeed presents with a relatively uniform clinical disease with pruritus as the predominant and initial sign, accompanied by behavioural and later locomotor changes as the disease progresses, and only rarely deficits of the menace response. the two arq/arq suffolk sheep inoculated intracerebrally displayed pruritus in a similar fashion to the bse cases infected orally or naturally. a temporal distribution in the display of clinical signs has also been shown in sheep scrapie, with an increased frequency of altered mental status, pruritus with wool loss, teeth grinding, head tremor, ataxia, loss of bodily condition and an absent menace response as the disease progresses. our findings suggest that the clinical presentation of bse in sheep of the susceptible or affected genotypes is largely influenced by the strain rather than breed, prp genotype or route of inoculation although the vast majority of bse cases were orally challenged arq/arq romney or suffolk sheep. more cases in other breeds or genotypes including orally inoculated vrq/vrq sheep need to be studied. natural infection, although only described in two cases in an experimental flock, did present with similar clinical signs and incubation periods. no other classical scrapie strain has yet been found in sheep with similar uniform neuropathological, molecular and biological characteristics as bse. clinical cases of atypical scrapie, which may be caused by a single strain based on the neuropathological and molecular characteristics, display predominantly behavioural and locomotor abnormalities but only very limited cases have been studied so far. well-characterised scrapie isolates, such as sspb/, produce a disease with variable clinical presentation in which pruritus and ataxia are displayed inconsistently in sheep of different breeds and genotypes inoculated subcutaneously. the variation seen in the neuroanatomical distribution of vacuolar changes and prpd accumulation in the brain of sheep with scrapie is believed to be affected not only by the scrapie strain but also by sheep breed and host genotype. differentiation of ovine bse from scrapie may not be possible based on clinical signs but our findings suggest that a scrapie suspect without signs of pruritus, such as the majority of atypical scrapie cases, is unlikely to have bse.  oral bse infection of romney and suffolk arq/arq sheep causes a uniform disease with early pruritus followed by behavioural changes and movement disorders as the disease progresses. identical findings in a limited number of additional bse cases described here suggest that the clinical features are independent of breed, genotype or route of inoculation. it is hypothesised that the clinical presentation in sheep is predominantly influenced by the tse strain. screening susceptible sheep for bse may be done efficiently by regular monitoring of their weights and testing them for a scratch response. 
clinical signs that were displayed significantly earlier in bse positive cases compared to negative cases were behavioural changes, pruritic behaviour, a positive scratch test, alopecia, skin lesions, teeth grinding, tremor, ataxia, loss of weight and loss of body condition. testing of the response to scratching along the dorsum of a sheep proved to be a good indicator of clinical disease with a test sensitivity of 85% and specificity of 98% and usually coincided with weight loss. our results suggest that bse in sheep presents with relatively uniform clinical signs, with pruritus of increased severity and abnormalities in behaviour or movement as the disease progressed. the study aimed to describe the clinical signs in sheep infected with the bse agent throughout its clinical course to determine whether the clinical signs were as variable as described for classical scrapie in sheep. the clinical signs were compared to bse-negative sheep to assess if disease-specific clinical markers exist. affected sheep belonged to five different breeds and three different genotypes. the variability in the clinical or pathological presentation of transmissible spongiform encephalopathies in sheep, such as scrapie and bovine spongiform encephalopathy, has been attributed to prion protein genotype, strain, breed, clinical duration, dose, route and type of inoculum and the age at infection. based on the studied sheep, these clinical features appear to be independent of breed, affected genotype, dose, route of inoculation and whether bse was passed into sheep from cattle or from other sheep, suggesting that the clinical phenotype of bse is influenced by the tse strain more than by other factors. the clinical phenotype of bse in the genotypes and breed studied was indistinguishable from that described for classical scrapie cases. pruritus was present in bse positive sheep; the remaining six were judged to be pre-clinically infected.
 the variability in the clinical or pathological presentation of transmissible spongiform encephalopathies in sheep, such as scrapie and bovine spongiform encephalopathy, has been attributed to prion protein genotype, strain, breed, clinical duration, dose, route and type of inoculum and the age at infection. the study aimed to describe the clinical signs in sheep infected with the bse agent throughout its clinical course to determine whether the clinical signs were as variable as described for classical scrapie in sheep. the clinical signs were compared to bse-negative sheep to assess if disease-specific clinical markers exist.  forty-seven of sheep, which comprised challenged sheep and undosed controls, were positive for bse. affected sheep belonged to five different breeds and three different genotypes. none of the controls or bse exposed sheep with arr alleles were positive. pruritus was present in bse positive sheep; the remaining six were judged to be pre-clinically infected. testing of the response to scratching along the dorsum of a sheep proved to be a good indicator of clinical disease with a test sensitivity of 85% and specificity of 98% and usually coincided with weight loss. clinical signs that were displayed significantly earlier in bse positive cases compared to negative cases were behavioural changes, pruritic behaviour, a positive scratch test, alopecia, skin lesions, teeth grinding, tremor, ataxia, loss of weight and loss of body condition. the frequency and severity of each specific clinical sign usually increased with the progression of disease over a period of 16– weeks.  our results suggest that bse in sheep presents with relatively uniform clinical signs, with pruritus of increased severity and abnormalities in behaviour or movement as the disease progressed. based on the studied sheep, these clinical features appear to be independent of breed, affected genotype, dose, route of inoculation and whether bse was passed into sheep from cattle or from other sheep, suggesting that the clinical phenotype of bse is influenced by the tse strain more than by other factors. the clinical phenotype of bse in the genotypes and breed studied was indistinguishable from that described for classical scrapie cases. 