Clinical signs, histopathology and genetics of experimental transmission of BSE and natural scrapie to sheep and goats

This paper compares the dinical signs, histopathology, detection of PrPSc protein and PrP genetics of the transmission of BSE to sheep and goats, with the effects of the transmission of natural scrapie from a brain homogenate from a single sheep. After intracerebral and oral inoculations there were similarities in the clinical signs due to the two sources of infection, but there were differences in pathology at the end stage of disease and in the genotypes of the sheep which succumbed to the challenges. The incubation period of BSE was associated with the sheep PrP codon 171 genotype, but the natural scrapie source, despite inducing disease only in known susceptible genotypes, showed no clear association with PrP genotype.     

疯牛病和羊痒病Western blotting检测方法的建立

以朊蛋白单抗AH6和碱性磷酸酶标记的马抗鼠酶标二抗建立了疯牛病和羊痒病的Western blotting检测方法。对Western blotting各种反应条件进行摸索。并确定了最佳工作条件，结果表明：匀浆缓冲液为RIPA时的最佳反应条件包括浓缩胶电泳电压为恒压90V，分离胶电泳电压为恒压160V，转印的最佳电压和时间为恒压100V1．5h；封闭液为3％BSA时，封闭15min，封闭效果最好；AH6的最佳稀释度为1：4000。4℃下孵育过夜。马抗鼠二抗的最佳稀释度1：1000，室温下孵育30min。采用已确立的反应条件对样品进行检测并与Prionics-Check WESTERN进口试剂盒的检测结果比较，发现其敏感性为100％，特异性为99．4％．与进口试剂盒（100％，100％）无显著差异，这为国产试剂盒研制提供了条件。     

Selection for scrapie resistance and simultaneous restriction of inbreeding in the rare sheep breed "Mergellander"

Scrapie is a fatal infectious neurodegenerative disease for which susceptibility is associated with polymorphisms in the ovine prion protein (PrP) gene. Scrapie-eradication programmes are based on eliminating the susceptible VRQ allele and/or breeding for the resistant ARR allele. In rare breeds or breeds with a low frequency of the ARR allele this can lead to unacceptably high inbreeding rates with associated increased risk of genetic defects and inbreeding depression. The conservation status of populations with inbreeding rates (DeltaF) above 1% is considered critical. In the Dutch rare sheep breed the Mergellander animals carrying ARR alleles are closely related to one another, and could reach 1.53% when only ARR/ARR animals are used as parents. Inbreeding rates can be reduced by selecting the set of parents according to their average co-ancestry. We minimised inbreeding rates by calculating the optimal contribution of each ram and selection of ewes. This resulted in inbreeding rates of -0.17% with exclusive use of homozygous ARR rams, and -0.38% if use of heterozygous rams was allowed as well. Thus sophisticated breeding programs can prevent unacceptably high inbreeding rates when breeding for scrapie resistance.     

Sensitivity and specificity of a commercial BSE kit for the detection of ovine scrapie

To examine the sensitivity of a commercially available bovine spongiform encephalopathy (BSE) kit (NippIBL) for the detection of ovine scrapie, 50 scrapie‐positive ovine samples from the UK, and 54 scrapie‐negative ovine samples from Japan were obtain and tested using this kit. The sensitivity and specificity of NippIBL for ovine samples were 96% and 100%, respectively. The detection limit of the abnormal isoform of prion protein (PrP^Sc) of NippIBL was examined using diluted scrapie‐positive samples. The sensitivity of NippIBL to ovine scrapie was 3–10 times superior to that of another commercial BSE diagnosis kit. Thus, the NippIBL kit proved more effective for the detection of ovine scrapie.     

Atypical/Nor98 scrapie: properties of the agent, genetics, and epidemiology

Atypical/Nor98 scrapie cases in sheep were diagnosed for the first time in Norway in 1998. They are now identified in small ruminants in most European countries and represent an increasingly large proportion of the scrapie cases diagnosed in Europe. Atypical/Nor98 scrapie isolates have shown to be experimentally transmissible into transgenic mice and sheep but the properties of the TSE agent involved, like its biological and biochemical features, are so clearly distinct from the agent involved in classical scrapie that they have provided a challenging diagnostic for many years. No strain diversity has yet been identified among the atypical/Nor98 scrapie sample cases. The genetic predisposition of the sheep affected by atypical/Nor98 scrapie is almost inverted compared to classical scrapie, and the exact origin of this sporadic TSE strain is still speculative, but a spontaneous, non-contagious origin, like sporadic Creutzfeldt-Jakob disease in humans, can not be excluded. Further transmission and epidemiological studies are needed to better address this hypothesis.     

Natural scrapie in British sheep: breeds, ages and PrP gene polymorphisms.

One hundred and sixty-seven sheep of 32 breeds and crossbreeds affected by natural scrapie throughout Britain were tested for the presence of restriction fragment length polymorphisms of the PrP gene observed when their DNA was digested with EcoRI or HindIII. These polymorphisms have already been associated with different susceptibilities to experimental scrapie (controlled by alleles of the Sip gene) in a flock of Cheviot sheep. In two studies 86 to 92 per cent of the sheep were found to carry the PrP gene EcoRI fragment e1 which is associated with high susceptibility (or the sA allele of Sip) to experimental scrapie. The PrP gene HindIII genotypes of the natural scrapie sheep were not apparently associated with differences in susceptibility to scrapie. There was no link between the polymorphisms and the age or breed of the affected sheep.     

Consequences of BSE/TSE for the clinical diagnostic in cattle and sheep

The first case of Bovine Spongiform Encephalopathy (BSE) in Germany induced a profound irritation not only of the consumers but also of the farmers and the veterinarians in Germany. The following BSE-crisis accelerated the structural changes in Beef and Dairy industries. The analysis of the detected BSE-cases of the last years in Germany and Switzerland shows that the sensitivity of BSE-tests is much higher in clinically preselected BSE-suspected cases compared to BSE-tests in normal slaughter cattle. Therefore it is necessary for an effective battle against BSE that clinical cases of CNS-diseases and disturbances of the locomotion especially in cows around partus should be clarified etiologically. If intra vitam the etiology of the disease can not be diagnosed by clinical and laboratory investigation of blood, urine and liquor samples and the therapy induces no improvement, the case has to be notified at the local veterinary officer. In the same way cases of Scrapie in sheep have to be handled. In sheep genotyping gives the opportunity to select for Scrapie resistance. In contrary to cattle with BSE Scrapie can be diagnosed intra vitam already before clinical symptoms occur by immunohistochemical investigations for Scrapie prion protein of tonsillar biopsies. These two diagnostic tools open new ways for fighting against Transmissible Spongiform Encephalopathies in sheep.     

Tono-Pen XL calibration curves for cats, cows and sheep

The objective of this study was to provide calibration curves for correcting intraocular pressure (IOP) measurements obtained using the Tono-Pen XL tonometer in cats, cows and sheep. Twelve eyes from 9 cats, 13 eyes from 7 cows, 10 eyes from 5 sheep were used. The anterior chamber of the eye was cannulated in vivo, in situ (immediately post mortem) or ex vivo with a fine needle and IOP was varied from 10 to 90 mmHg in steps of 10 mmHg by adjusting the height of a saline reservoir connected to the needle. For each pressure setting, several readings of IOP were made using the tonometer. The relationship between Tono-Pen reading and manometer setting was linear over the full range of measurement. However, the slope of the data regression line deviated significantly from 1 and indicated that the instrument systematically underestimated IOP. For cats the average slope was 0.62 and for cows and sheep it was 0.72 and 0.69, respectively. For the latter animals, the regression line also had a nonzero intercept of approximately 4.5 mmHg. Similar results were obtained from in vivo and ex vivo eyes and with different Tono-Pen XL tonometers. Although developed for use on humans, the Tono-Pen XL can provide reproducible and accurate measurement of IOP in cats, cows and sheep when suitably calibrated by manometry. The calibration curves provided here, and by implication those reported for other animals using this tonometer, differ in slope from those measured with earlier models of the Tono-Pen. The reproducibility of the curves we obtained implies that they can be used to correct IOP readings from the Tono-Pen XL when manometry is not possible.     

Susceptibility of transgenic mice expressing chimeric sheep,bovine and human PrP genes to sheep scrapie

The use of Transgenic (Tg) mice expressing chimeric sheep/mouse (Sh/Mo) prion protein (PrP) and chimeric bovine/mouse (Bo/Mo) PrP genes was evaluated as a sheep scrapie model. We also investigated the potential for the transmission of sheep scrapie to a human/mouse (Hu/Mo) PrP Tg mouse line. The Sh/Mo PrP and Bo/Mo PrP Tg Prnp(+/+) or Prnp(0/0) mouse lines were inoculated intracerebrally with brain homogenates from three sheep with natural scrapie (KU, Y5 or S2). Incubation periods were slightly shorter in Sh/Mo PrP Tg Prnp(+/+), than in non-Tg mice inoculated with KU brain homogenate. In contrast, the incubation period was significantly prolonged (p<0.05) in Bo/Mo PrP Tg Prnp(+/+) mice inoculated with KU brain homogenate. The incubation period was significantly longer in all Tg Prnp(+/+) and Prnp(0/0), than in non-Tg mice (p<0.01) inoculated withY5 brain homogenate. None of the Tg Prnp(0/0) mice inoculated with S2 brain homogenate developed clinical signs and PrP(Sc) was undetectable in their brains. These results suggested that expression of the Sh/Mo PrP or Bo/Mo PrP transgenes does not confer susceptibility to sheep prions upon mice, and thus none of the Tg mouse lines could be a suitable model of sheep scrapie. Hu/Mo PrP Tg Prnp(0/0) mice inoculated with natural and experimental scrapie or mouse prions did not develop clinical signs of scrapie and PrP(Sc) was undetectable. These results suggested that neither sheep nor mouse strains of scrapie are highly transmissible to humans.     

Recognition of the Nor98 variant of scrapie in the Swedish sheep population.

Within the framework of the active surveillance for transmissible spongiform encephalopathies in sheep in Sweden, 4 cases of the atypical form of scrapie, Nor98, were identified during 2003. Nor98 is a recently recognized and poorly understood variant of scrapie, first described in Norway. The cases were positive by the rapid test (enzyme-linked immunosorbent assay). Immunohistochemical staining showed diffuse thin-granular staining of the cerebellar cortex. Western immunoblotting analysis of specimens of brain stem and cerebellum showed a light band of approximately 12 kDa. Typical scrapie was ruled out based on the confirmatory testing. The affected ewes were from 4 different flocks. They were between 7 and 9 years old. Two were of the ARQ/ARQ genotype, 1 ARR/ARQ, and 1 ARR/AHQ. Two ewes had shown ataxia, and the other 2 had no clinical signs. Whole-flock slaughter was applied, and testing of the flock mates did not reveal additional cases. Nor98 differs from typical scrapie in its epidemiology, frequency of genotypes of sheep affected, clinical signs, microscopic lesions, distribution of scrapie prion protein in the brain, and characteristics of the immunostaining and immunoblotting profiles.     

Experimental transmission of sheep scrapie by intracerebral and oral routes to genetically susceptible Suffolk sheep in the United States.

Scrapie is a naturally occurring fatal neurodegenerative disease of sheep and goats. Susceptibility to the disease is partly dependent on the genetic makeup of the host. This study documents clinicopathological findings and the distribution of abnormal prion proteins (PrPres) by immunohistochemical and Western blot techniques, in tissues of genetically susceptible sheep inoculated with US sheep scrapie agents. Four-month-old Suffolk lambs (QQ or HQ at codon 171) were inoculated (5 intracerebrally and 19 orally) with an inoculum (#13-7) consisting of a pool of scrapie-affected sheep brains. Intracerebrally inoculated animals were euthanized when advanced clinical signs of scrapie were observed. Orally inoculated animals were euthanized at predetermined time points (4, 9, 12, 15, and 21 months postinoculation [PI]) and thereafter when the animals had terminal signs of disease. All intracerebrally inoculated animals exhibited clinical signs of scrapie and were euthanized between 13 and 24 months PI. Spongiform lesions in the brains and PrPres deposits in central nervous system and lymphoid tissues were present in these sheep. In orally inoculated sheep, clinical signs of scrapie were seen between 27 and 43 months PI in 5/9 animals. The earliest detectable PrPres was observed in brainstem and lymphoid tissues of a clinically normal, orally inoculated sheep at 15 months PI. Three of the 4 clinically normal sheep were positive at 15, 20, and 49 months PI by PrPres immunohistochemistry.     

Comparative pharmacokinetics of enrofloxacin in mice, rats, rabbits, sheep, and cows.

OBJECTIVE: To compare pharmacokinetic variables of enrofloxacin (ENR) after IV administration in mice, rats, rabbits, sheep, and cows and to perform allometric analysis of ENR. ANIMALS: 47 mice, 5 rats, 5 rabbits, 5 sheep, and 5 cows. PROCEDURE: Serially obtained plasma samples were assayed for ENR concentration, using high-performance liquid chromatography. In vitro plasma protein binding was determined by ultrafiltration. Plasma ENR concentration versus time curves were fitted by use of nonlinear least-squared regression analysis. Pharmacokinetic variables were correlated further with body weight. RESULTS: In all species studied, the best fit was obtained for a two-compartment open model; ENR half-life ranged from 89 minutes in mice to 169 minutes in cows. Volume of distribution was large in all species studied, with values ranging from 10.5 L/kg in mice to 1.5 L/kg in sheep. Body clearance ranged from 68.1 ml/min/kg for mice to 4.6 ml/min/kg for sheep. Unbound ENR was found to be (mean +/- SD) 58+/-2, 50+/-6, 50+/-2, 31+/-2, and 40+/-3% in plasma of mice, rats, rabbits, sheep, and cows, respectively. The only pharmacokinetic variables that could be correlated with body weight were elimination half-life, clearance, and volume of distribution. Allometric exponents denoting proportionality of half-life, body clearance, and volume of distribution with body weight were 0.06, 0.82, and 0.90, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: An allometric approach could provide a suitable method for determining a scale for ENR pharmacokinetics among various mammalian species. This would faciliatate the administration of appropriate doses of ENR to all animals.     

Breeding programmes for TSE resistance in British sheep. II. Assessing the impact on the prevalence and incidence of scrapie

By establishing a breeding programme for transmissible spongiform encephalopathie (TSE) resistance, there are plans to eradicate sheep TSEs from member states of the European Union (EU). In this paper, we used a simple age- and genotype-structured model to assess the impact of four breeding strategies on the prevalence and incidence of scrapie in the British sheep flock. The strategies ranged from the minimum EU requirements to compulsory implementation of the current National Scrapie Plan for Great Britain (NSP). All four strategies were predicted to reduce the prevalence and incidence of disease, though there was likely to be a delay of several years between the implementation of a breeding programme and the reduction in incidence. There were differences in the efficacy of the strategies, with the most stringent resulting in the greatest reduction in prevalence and incidence. However, the magnitude of the differences was not great, largely because all four strategies eliminated the VRQ allele, which is associated with a markedly higher risk of disease than any of the other alleles. Sensitivity analyses indicated that the model results were robust to selection bias when estimating the risk of infection; and that the efficacy of a breeding programme was unlikely to be compromised, unless the risk of infection is substantially underestimated by data on clinical disease.     

Swiss scrapie surveillance. I. Clinical aspects of neurological diseases in sheep and goats

Small ruminants infected with scrapie show a large range of often unspecific clinical symptoms. The most-often described signs, locomotion, sensibility and behavioural disorders and emaciation, rarely occur together, and cases have been described in which only one of those signs was detectable.Thus, formulating a well-circumscribed definition of a clinical suspect case is difficult. Most animals with CNS-effecting diseases such as listeriosis, polioencephalomacia, cerebrospinal nematidiasis and enterotoxemia will, in a thorough neurological examination, show at least some scrapie-like symptoms. Among the 22 neurological field cases examined in this study, a goat with cerebral gliomatosis and hair lice showed the closest similarity to clinical scrapie. The unilateral deficiency of the cerebral nerves has potential as an clinical exclusion criterion for scrapie. However, the laboratory confirmation--or exclusion--of scrapie remains important. It thus needs to be realized that a consistent and thorough examination of neurologically diseased small ruminants (including fallen stock) is the backbone of a good surveillance system for these diseases. This should be a motivation for submitting adult sheep and goats for neuropathological examination.     

Adaptation and evaluation of a rapid test for the diagnosis of sheep scrapie in samples of rectal mucosa.

In recent publications, it was shown that disease-associated prion protein (PrP(d)) accumulates in the lymphoid tissue of the rectal mucosa of a high proportion of scrapie-infected sheep at clinical and preclinical stages, regardless of several host factors; PrP(d) can also be detected in biopsy specimens of rectal mucosa, with an increased probability proportional to age or incubation period and with an efficiency almost identical to that of tonsil biopsies. Rectal biopsies have the advantages of providing higher numbers of lymphoid follicles and of being simpler to perform, which makes them suitable for scrapie screening in the field. In biopsy samples, PrP(d) could be demonstrated by immunohistochemical (IHC) and Western immunoblotting methods, and the purpose of the present study was to optimize and evaluate a "rapid test" for the diagnosis of scrapie in rectal biopsy samples. The HerdChek CWD (chronic wasting disease) antigen EIA (enzyme immunoassay) test was chosen and, once optimized, provided specificity and sensitivity figures of 99.2% and 93.5%, respectively, compared with IHC results in the same samples obtained at a postmortem. The sensitivity of the assay increased from 82.1%, when a single rectal mucosa sample was tested to 99.4% for those sheep in which 3 or more samples were analyzed. Similarly, sensitivity values of the HerdChek CWD antigen EIA test on biopsy samples increased from 95% to 100% for sheep subjected to 1 or 2 sequential biopsies 4 months apart, respectively. Thus, a preclinical diagnosis of scrapie in live sheep can be achieved by a combination of a simple sampling procedure, which can be repeated several times with no detrimental effect for the animals, and a rapid and efficient laboratory method.     

Discriminant value of blood and urinary corticoids for the diagnosis of scrapie in live sheep

The mean (sd) concentration of plasma 20beta-dihydrocortisol in 126 scrapie-affected sheep was 5-5 (7.0) ng/ml compared with 1.1 (0.7) ng/ml in 52 healthy sheep. The mean (sd) concentration of creatinine in the urine of 93 scrapie-affected sheep was 2.43 (1.56) microg/ml compared with 0.94 (0.86) pg/ml in 49 healthy sheep and 1.10 (0-95) pg/ml in 25 sheep with other diseases. These discriminant analyses carried out on healthy and scrapie-affected sheep showed that plasma 20beta-dihydrocortisol and urinary creatinine were the best predictors of the disease, and classified correctly 98 per cent of healthy sheep and 82 per cent of scrapie-affected sheep.     

Swiss scrapie surveillance. II. Epidemiologic aspects of the detection of neurological diseases in sheep and goats

Monitoring of transmissible spongiform encephalopathy (TSE) in Swiss sheep and goats is based on the examination of animals from different sources. In this study, frequencies and proportions of the different diagnoses were compared between routinely submitted sheep and goats, notified scrapie suspects as well as fallen stock. Meningitis/ encephalitis cases were significantly more frequent (OR = 2.2) in the scrapie suspect group when compared to the routine submissions. Metabolic-toxic encephalopathy was seen more frequently within the fallen stock. Rare neurological diagnoses were more frequent among scrapie suspects and routine submissions when compared to fallen stock. Listeriosis was diagnosed equally frequent among the scrapie suspects and routine submissions but less frequent in fallen stock. Scrapie prevalence among the fallen stock and the routine submissions was 0 (zero), with 95% certainty that prevalence is < 1%. The examined animals are representative for most of the Swiss regions with considerable sheep and goat production. Continuation of the detailed neuropathological examination of small ruminants from these three groups, substituted by actively testing a sufficiently large sample of fallen stock and possibly also healthy-slaughtered adult sheep and goats for transmissible spongiform encephalopathies would ensure a good surveillance within the small ruminant population.     

Effectiveness of capillary electrophoresis fluoroimmunoassay of blood PrpSc for evaluation of scrapie pathogenesis in sheep.

Management of prion diseases in livestock would benefit greatly from availability of a validated blood test. A promising immunocapillary electrophoresis technique (also known as capillary electrophoresis fluoroimmunoassay) to detect abnormal prion protein in blood from live sheep is evaluated here. Capillary electrophoresis fluoroimmunoassay was applied to analysis of extracted blood from scrapie-exposed sheep (n = 87; 347 samples) at various stages of incubation, and to control sheep (n = 194; 489 samples). Overall, test values for the control and test populations were not significantly different, and a similar proportion of control (7%) and test (10%) sheep were classified as positive. Over 2-3 month intervals from birth until clinical disease, test specificity and sensitivity ranged from 66.7% to 100% and 0% to 66.7%, respectively, indicating poor diagnostic performance at all stages of pathogenesis. In routine application, in its present form, the capillary electrophoresis fluoroimmunoassay procedure proved to be insufficiently robust for use as a blood test for scrapie diagnosis.