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1.
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.  相似文献   

2.
Samples of tissue from the central nervous system (cns), the lymphoreticular system (lrs) and the rectal mucosa of a large number of scrapie-exposed sheep, with and without signs of clinical disease, were examined immunohistochemically for evidence of disease-associated prion protein (PrP(d)). The rectal mucosa has received almost no attention so far in scrapie diagnosis, despite its abundant rectoanal mucosa-associated lymphoid tissue, and its accessibility. The scrapie-confirmed cases included 244 with clinical disease, of which 237 (97.1 per cent) were positive in the rectal mucosa, and 121 apparently healthy sheep, of which 104 (86 per cent) were positive in the rectal mucosa. PrP(d) was detected in 86.4 to 91.5 per cent of the other lrs tissues of the healthy sheep examined and in 77.7 per cent of their cns tissues. The stage of infection, therefore, affected the probability of a positive result in the rectal mucosa, whereas the breed, PrP genotype, age and sex had little or no independent effect. Accumulations of PrP(d) were observed in the rectal mucosa and other lrs tissues of vrq/arr sheep with preclinical and clinical scrapie, albeit with a lower frequency and magnitude than in sheep of other PrP genotypes. Western immunoblotting analyses of samples of rectal mucosa gave the characteristic PrP glycoprofile, with a sensitivity similar to that of immunohistochemistry.  相似文献   

3.
Tissue samples were collected postmortem from 126 sheep at five lymphoreticular sites by different techniques. The three most successful combinations of sites and techniques were: the third eyelids, using a forceps and scissors, which provided a mean (se) of 5.32 (0.70) lymphoid follicles per 5 microm tissue section, a mandibular lymph node, using a Biopty gun, which gave 1.19 (0.26) lymphoid follicles per 5 microm tissue section, and tonsil, using a biopsy forceps, which gave 1.14 (0.27) lymphoid follicles per 5 microm tissue section. These three techniques were repeated once a month for five months on five sheep under general anaesthesia, and their clinical effects were compared with five control sheep which were restrained and anaesthetised in the same way but from which no biopsies were taken. Most lymphoid follicles (3.47 [0.58] per 5 pm tissue section) were obtained by using the third eyelid biopsy technique. There were no clinical side effects associated with the biopsy procedure. There were increases in the plasma concentration of cortisol in all the animals, suggesting that the restraint and anaesthesia were more stressful than the biopsy procedure.  相似文献   

4.
The ovaries of 59 pluriparous cows of unknown reproductive history were palpated, scanned and dissected on the day of slaughter to compare the accuracy of rectal palpation and transvaginal ultrasonography with a 5 MHz linear array for the detection of corpora lutea and follicles. The rectal palpation was first carried out to judge the presence of follicles of more than 5 mm diameter, and corpora lutea which were classified as young (days 1 to 4), mid-cycle (days 5 to 16) or old (days 17 to 21) according to morphological criteria. The cows were then examined for follicles and corpora lutea by ultrasonography and the corpora lutea were again classified directly as young, mid-cycle or old according to their appearance. The cows were then slaughtered, their ovaries dissected, and the follicles over 5 mm in diameter were counted and the corpora lutea were classified in the above mentioned age categories. For the detection of a mid-cycle corpus luteum the sensitivity and predictive value of rectal palpation were, respectively, 83.3 per cent and 73.2 per cent and for ultrasonography the sensitivity and predictive value were 80.6 per cent and 85.3 per cent, respectively. However, both techniques were inaccurate for the detection of young and old corpora lutea. For detecting follicles ultrasonography was a significantly better method than rectal palpation. Ultrasonography detected 95 per cent of follicles larger than 10 mm whereas rectal palpation detected only 71 per cent of these follicles. Both techniques failed with follicles 5 to 10 mm in diameter; only 21.5 per cent were detected by rectal palpation and 34.3 per cent by ultrasonography.  相似文献   

5.
Since scrapie and bovine spongiform encephalopathy (BSE) in sheep are clinicopathologically indistinguishable, BSE in sheep may have been misdiagnosed as scrapie. Disease-specific prion protein (PrP(d)) patterns in archival tissues of 38 Irish ARQ/ARQ sheep diagnosed as scrapie-affected were compared to those in four Dutch BSE-challenged sheep. When medulla oblongata was immunolabelled with an antibody directed against amino acids 93-99 of ovine prion protein (ovPrP), intraneuronal PrP(d) was apparent in all 38 Irish sheep but was absent in BSE-challenged sheep. When lymphoid follicles were immunolabelled with antibodies directed against amino acids 93-106 of ovPrP, granule clusters of PrP(d) were seen in 34 of the 38 Irish sheep. Follicles of the remaining four archive sheep contained either no PrP(d) or single PrP(d) granules, similar to follicles from BSE-challenged sheep. Based on the medulla results, none of the archival cases had BSE-derived disease. The identification of some scrapie sheep with little or no intrafollicular PrP(d) suggests that this technique may be limited in discriminating between the two diseases.  相似文献   

6.
The objective of this study was to investigate and estimate the associations of the ovine prion protein (PrP) genotypes with a wide range of performance traits in Scottish Blackface lambs. Performance records of up to 7,138 sheep of known PrP genotypes born from 1999 to 2004 in 2 experimental farms were utilized. Performance traits studied were BW at birth, marking (when the sheep were identified with permanent ear tags at an average age of 52 d), and weaning (average age of 107 d); slaughter traits (BW at slaughter, slaughter age, carcass weight, and carcass conformation); ultrasonic muscle and fat depths; and computerized tomography-predicted carcass composition and carcass yield at weaning. Different linear mixed models, including random, direct animal effect, and up to 3 maternal effects (genetic, permanent, and temporary environmental) were used for the different traits. The PrP genotype was included in the model as a fixed effect, along with other fixed factors with significant effects (P < 0.05). Five separate analyses were carried out for each trait, differing in the method of PrP genotypic classification. The first analysis was based on classifying the sheep into categories according to all 9 available PrP genotypes. In the other 4 analyses, sheep were categorized according to the number of each PrP allele carried. Results showed that there were no significant differences between PrP genotypes for any of the performance traits studied when all 9 genotypes were compared (first analysis). Similarly, performance of the lambs did not significantly differ between genotypes with different numbers of ARR copies. However, there were significant variations in a few traits with respect to the number of ARQ, AHQ, and VRQ alleles carried. Heterozygous lambs for the AHQ or the ARQ allele were significantly heavier at some ages than lambs of the other genotypes. Lambs carrying the VRQ allele required approximately 10 d longer finishing time (P = 0.01) and yielded carcasses approximately 0.5 kg heavier (P = 0.03) compared with noncarriers. The few significant associations found do not have a negative influence on performance when selecting against the most susceptible PrP allele (VRQ) or in favor of the most resistant one (ARR). Overall, there were no major associations of PrP genotypes with most lamb performance traits in Scottish Blackface sheep.  相似文献   

7.
There have been no reports of natural scrapie in Irish Blackface Mountain (BM) sheep which account for approximately 16% of the Irish national sheep flock. The aim of this study was to determine if Irish BM sheep had unusual clinical and/or pathological features of scrapie which would account for failure to diagnose the disease in this breed. BM (n=7), Texel (n=3) and Suffolk sheep (n=1) of scrapie-susceptible PrP genotypes (ARQ/ARQ and VRQ/ARQ) were orally challenged with scrapie-infected brain inoculum. The incubation period, clinical signs, pathology and distribution of disease specific prion protein (PrP(d)) in scrapie-affected BM sheep were similar to scrapie in the Texel and Suffolk sheep. It was concluded that there was no evidence to suggest that scrapie in BM sheep differs clinicopathologically from scrapie in other breeds of sheep.  相似文献   

8.
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.  相似文献   

9.
Two new PCR-based methods were developed to decode prion protein (PrP) gene polymorphisms at codons 136, 154 and 171: a PCR-restriction fragment length polymorphism (RFLP) analysis consisting of two PCR reactions followed by three enzymatic digestions, and a real-time PCR consisting of four reactions with seven fluorogenic probes. Both methods were used to study the distribution of PrP gene polymorphisms in a representative sample (1297 animals) of the populations of the two native breeds of sheep of the Spanish Basque Country, Latxa and Carranzana. Fourteen genotypes were found in the Latxa breed, in which ARQ/ARQ was the genotype most frequently observed (49.3 per cent), followed by ARR/ARQ (32.6 per cent) and ARQ/ARH (5.8 per cent). The genotype associated with the highest resistance to scrapie (ARR/ARR) was present in 5 per cent of the animals analysed. Similar results were observed in the Carranzana sheep.  相似文献   

10.
AIMS: To estimate the number of cases of scrapie that would occur in sheep of different prion protein (PrP) genotypes if scrapie was to become established in New Zealand, and to compare the performance of two commercially available, rapid ELISA kits using ovine retro-pharyngeal lymph nodes (RLN) from non-infected and infected sheep of different PrP genotypes.

METHODS: Using published data on the distribution of PrP genotypes within the New Zealand sheep flock and the prevalence of cases of scrapie in these genotypes in the United Kingdom, the annual expected number of cases of scrapie per genotype was estimated, should scrapie become established in New Zealand, assuming a total population of 28 million sheep. A non-infected panel of RLN was collected from 737 sheep from New Zealand that had been culled, found in extremis or died. Brain stem samples were also collected from 131 of these sheep. A second panel of infected samples comprised 218 and 117 RLN from confirmed scrapie cases that had originated in Europe and the United States of America, respectively. All samples were screened using two commercial, rapid, transmissible spongiform encephalopathy ELISA kits: Bio-Rad TeSeE ELISA (ELISA-BR), and IDEXX HerdChek BSE-Scrapie AG Test (ELISA-ID).

RESULTS: If scrapie became established in New Zealand, an estimated 596 cases would occur per year; of these 234 (39%) and 271 (46%) would be in sheep carrying ARQ/ARQ and ARQ/VRQ PrP genotypes, respectively. For the non-infected samples from New Zealand the diagnostic specificity of both ELISA kits was 100%. When considering all infected samples, the diagnostic sensitivity was 70.4 (95% CI=65.3–75.3)% for ELISA-BR and 91.6 (95% CI=88.2–94.4)% for ELISA-ID. For the ARQ/ARQ genotype (n=195), sensitivity was 66.2% for ELISA-BR and 90.8% for ELISA-ID, and for the ARQ/VRQ genotype (n=107), sensitivity was 81.3% for ELISA-BR and 98.1% for ELISA-ID.

CONCLUSIONS: In this study, the ELISA-ID kit demonstrated a higher diagnostic sensitivity for detecting scrapie in samples of RLN from sheep carrying scrapie-susceptible PrP genotypes than the ELISA-BR kit at comparable diagnostic specificity.

CLINICAL RELEVANCE: The diagnostic performance of the ELISA-ID kit using ovine RLN merits the consideration of including this assay in the national scrapie surveillance programme in New Zealand.  相似文献   

11.
This study investigated whether the abnormal prion protein (PrP(Sc)) in tissues from sheep with scrapie would be destroyed by composting. Tissues from sheep naturally infected with scrapie were placed within fiberglass mesh bags and buried in compost piles for 108 d in experiment 1 or 148 d in experiment 2. The temperature in the compost piles rose quickly; it was above 60 degrees C for about 2 wk and then slowly declined to the ambient temperature. Before composting, PrPSc was detected in all the tissues by Western blotting. In experiment 1, PrPsc was not detected after composting in the tissue remnants or the surrounding sawdust. In experiment 2, 1 of 5 specimens tested negative after composting, whereas PrP(Sc) was detected in the other 4 bags, though in reduced amounts compared with those before composting. Tissue weights were reduced during composting. Analysis of the tissue remnants for microbial 16S ribosomal DNA demonstrated that there were more diverse microbes involved in experiment 1 than in experiment 2 and that the guanine and cytosine content of the microbial 16S DNA was higher in the specimens of experiment 1 than in those of experiment 2, which suggests greater dominance of thermophilic microbes in experiment 1. These results indicate that composting may have value as a means for degrading PrP(Sc) in carcasses and other wastes.  相似文献   

12.
Maedi-visna, a multisystemic disease of adult sheep, was first described in Spain in 1984. To get an idea of the seroprevalence of the disease locally and to estimate the number of seropositive animals with lesions, samples of blood, lungs and mammary glands were taken from 124 randomly selected sheep killed in the main slaughterhouse of Zaragoza. In the agar gel immunodiffusion test, 74 (59.7 per cent) of the sheep were positive and 50 were negative. Among the 74 seropositive animals, 19 (25.6 per cent) had no lesions in any organ, 12 (16.2 per cent) had lesions in the lungs only, 15 (20.2 per cent) had lesions in the mammary glands and 28 (37.8 per cent) had lesions in both organs. In the lungs hyperplasia of lymphoid follicles was more evident than an interstitial infiltrate but in the mammary glands this relationship was not observed. Even when the lesions occurred in both organs, they did not show the expected proportion in terms of either type or severity. Among the 50 seronegative sheep, eight (16 per cent) showed maedi-like lesions, formed exclusively by the hyperplasia of lymphoid follicles.  相似文献   

13.
Susceptibility to clinical scrapie is associated with polymorphisms in the prion protein (PrP) gene. The ARR allele reduces susceptibility to clinical disease caused by all known strains of the transmissible spongiform encephalopathy (TSE) agents. For the economically important German breeds of sheep the PrP allele frequencies are well known, but this paper presents representative genotyping results for 1526 sheep from two smaller milk sheep breeds and 2446 sheep from 14 mostly indigenous land sheep breeds. The ARR allele was detected in each breed but the breed-specific ARR frequencies varied between 1 and 63 per cent. In small populations with a very low ARR frequency the ARR allele could be lost by genetic drift. A simulation study was therefore made to examine the effects of different breeding schemes in populations of different sizes on attempts to select for the ARR allele in an endangered population. In breeds in which no homozygous rams are available the breeding strategy would depend on the number of heterozygous rams, and the genotyping and selection of suitable breeding ewes would reduce the time required to achieve a highly resistant population. In general, in all the breeds a selection programme to achieve 99 per cent ARR homozygous genotypes would be feasible in six to nine generations, depending on the initial allele frequencies. In small populations the inbreeding rate may increase if no specific mating plans are developed by the breeding organisations.  相似文献   

14.
Scrapie is a natural transmissible spongiform encephalopathy (TSE) of sheep, infecting the animal via the gastrointestinal tract or the skin. This project tested the hypotheses that lymph-borne cells (especially dendritic cells) are crucial for the systemic dissemination of the infectious agent from the site of infection in the skin, that PrP genotype affects PrPSC association with dendritic cells and that PrPSC carriage by cells affects their expression of cytokines. Skin, of scrapie-susceptible VRQ/ARR and scrapie-resistant ARR/ARR PrP genotypes, was scarified with FITC-labelled PrPSC. Pseudoafferent lymphatic cannulation was then used to monitor the presence of FITC-PrPSC over time in different lymph cell populations and plasma in the draining afferent lymphatics. The major observation was that PrPSC did not associate significantly with any lymphocyte or dendritic cell population in the 5 days following PrPSC scarification. The only cells seen to associate with PrPSC were neutrophils. Furthermore, despite the quantity of PrPSC used for scarification being equivalent to a standard infectious dose (the VRQ/ARR sheep dying at approximately 260 days) the only PrP found in afferent lymph during the 0-5-day period was proteinase K sensitive (i.e. soluble PrPC). No differences were observed between the PrP genotypes. Analysis of the effects of PrPSC scarification of cellular cytokine mRNA expression (by a nuclease protection assay) showed raised levels of IL-1beta and IL-8 in the susceptible VRQ/ARR group and raised levels of IFNgamma in the resistant ARR/ARR animals.  相似文献   

15.
Prion protein gene polymorphisms in pedigree sheep in Ireland   总被引：4，自引：0，他引：4  
The development of clinical signs of scrapie in sheep has been linked to polymorphisms in the prion protein (PrP) gene. The most important polymorphisms appear to be at codons 136, 154 and 171. The objective of this study was to investigate polymorphisms at these codons in five native (Belclare, Galway, Wicklow Cheviot, Donegal Blackface Mountain and Mayo Blackface Mountain) and five imported (Texel, Bleu du Maine, Rouge de l'Ouest, Vendéen and Charollais) sheep breeds in Ireland. A total of 13 genotypes were found. The percentage of the most resistant genotype AA(136)RR(154)RR(171)varied from 1.8 per cent in the Vendéen breed, 3.1 per cent in Donegal Blackface Mountain, 10.0 per cent in Texel, 11.1 per cent in Wicklow Cheviot, 12.9 per cent in Belclare, 22.0 per cent in Charollais, 25.6 per cent in Mayo Blackface Mountain, 33.3 per cent in Galway, 46.4 per cent in Bleu du Maine to 62.5 per cent in Rouge de l'Ouest. The results indicate that a significant amount of variation exists between the breeds analysed in this study.  相似文献   

16.
A randomised sample of 2,809 apparently healthy sheep, 55 per cent of them less than 15 months of age, which were slaughtered for human consumption at abattoirs in Great Britain in 1997/98, was taken to establish the prevalence of scrapie infection. The medulla oblongata of each sheep was examined histopathologically at the level of the obex, and fresh brain tissue was examined for scrapie-associated fibrils (SAF) to establish whether there was evidence of scrapie. In addition, histological sections of the medulla from 500 of the sheep were immunostained with an antiserum to PrP, and the same technique was also applied to any animal found positive or inconclusive by the histological or SAF examinations. Any sheep which was positive by any of these diagnostic methods was also examined by Western immunoblotting, for the detection of the disease-specific protein PrP(Sc). A total of 2,798 sheep (99.6 per cent) were negative by all the methods applied. Ten animals were SAF-positive but negative by all the other methods, and in one animal there was immunohistochemical staining which could not be interpreted unequivocally as disease-specific. A mathematical model was used to estimate the prevalence of scrapie infection in the national slaughtered sheep population which would be consistent with these results. By this model, the absence of unequivocally substantiated cases of scrapie in the sample was consistent with a prevalence of infection in the slaughter population of up to 11 per cent.  相似文献   

17.
The usefulness of detecting the scrapie-associated fibrillar protein (PrP) in the lymphoreticular organs of sheep as a diagnostic tool was investigated. The PrP was detected by means of a rabbit-anti-sheep PrP polyclonal antibody by Western blot analysis. PrP was detected in samples from the central nervous system (CNS) of five of six sheep showing clinical signs of natural scrapie infection, in spleen samples from four of the six sheep and in lymph node samples taken from three of the sheep. PrP was detected in the spleen and lymph node samples, but not in the CNS samples from one of the six sheep that was clinically and histopathologically abnormal. This animal appeared to be in the early clinical stage of the disease. A total of 47 clinically normal sheep were examined for the presence of PrP. It was detected in spleen samples from three of the 47 sheep and in lymph node samples from three of the 39 sheep tested. Similarly, PrP was detected in a sample of lymph node obtained surgically from one of three experimentally infected sheep 14 months after inoculation. The PrP-positive sheep and one of the remaining PrP-negative sheep showed clinical signs of scrapie six and five months later respectively. One sheep euthanased 18 months after experimental infection was positive for PrP in the CNS, spleen and lymph node, but five other sheep which were killed or died two, eight, 16, 18 and 21 months after infection were negative or doubtful for the detection of PrP.  相似文献   

18.
OBJECTIVE: To evaluate a polymerase chain reaction-based assay for the detection of Mycobacterium avium subsp paratuberculosis in blood and liver biopsies from subclinically infected sheep. STUDY DESIGN: A direct PCR assay for the detection of M a paratuberculosis was applied to liver biopsy specimens and to samples of blood that were sequentially collected over 53 weeks from 14 sheep infected experimentally with the organism. RESULTS: Of 117 blood samples from the 14 experimentally infected sheep, two tested positive in the PCR assay. Both positive results were obtained in two subclinically infected sheep that had paratuberculosis later confirmed by histological examination at necropsy. However, the assay failed to detect the target DNA in samples of blood from five other sheep with histologically confirmed paratuberculosis. Similarly, the PCR assay on liver biopsy specimens collected 32 weeks after administration of M a paratuberculosis gave only two positive results, both of which were obtained in sheep with histological evidence of paratuberculosis. CONCLUSIONS: The PCR assay on blood and liver biopsies does not provide a useful method for the diagnosis of M a paratuberculosis infection in subclinically infected sheep.  相似文献   

19.
Scrapie and bovine spongiform encephalopathy (BSE) are both prion diseases affecting ruminants, and these diseases do not share the same public health concerns. Surveillance of the BSE agent in small ruminants has been a great challenge, and the recent identification of diverse prion diseases in ruminants has led to the development of new methods for strain typing. In our study, using immunohistochemistry (IHC), we assessed the distribution of PrP(d) in the brains of 2 experimentally BSE-infected sheep with the ARQ/ARQ genotype. Distribution of PrP(d) in the brain, from the spinal cord to the frontal cortex, was remarkably similar in the 2 sheep despite different inoculation routes and incubation periods. Comparatively, overall PrP(d) brain distribution, evaluated by IHC, in 19 scrapie cases with the ARQ/ARQ, ARQ/VRQ, and VRQ/VRQ genotypes, in some cases showed similarities to the experimentally BSE-infected sheep. There was no exclusive neuroanatomical site with a characteristic and specific PrP(d) type of accumulation induced by the BSE agent. However, a detailed analysis of the topography, types, and intensity of PrP(d) deposits in the frontal cortex, striatum, piriform cortex, hippocampus, mesencephalon, and cerebellum allowed the BSE-affected sheep group to be distinguished from the 19 scrapie cases analyzed in our study. These results strengthen and emphasize the potential interest of PrP(d) brain mapping to help in identifying prion strains in small ruminants.  相似文献   

20.
The frequencies of prion protein (PrP) genotypes were investigated in 15 scrapie-affected flocks in Great Britain. The flocks were heterogeneous in the frequencies of different genotypes and alleles, and in their age distributions. The median flock frequency of animals with VRQ-containing genotypes was 21 per cent (range 2 to 82 per cent, mean 25 per cent). The VRQ-containing and other non-ARR genotypes made up 11 to 82 per cent of a flock (median 46 per cent, mean 48 per cent). In comparison with data from the general population the scrapie-affected population had a lower frequency of the ARR/ARR genotype, and so of the ARR allele, and had a higher frequency of VRQ/non-ARR heterozygote genotypes, and thus of the VRQ allele.  相似文献   

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