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.     

BSE and scrapie diagnosis in Germany

The detection of pathological prion protein is considered as pathognomonic for the diagnosis of transmissible spongiform encephalopathies. According to the EU regulations cattle older than 30 months of age (Germany 24 months) and slaughtered for human consumption must be tested by using BSE rapid tests. Likewise must be fallen stock and clinically affected animals. This article gives an overview over the diagnostic hierarchy and organization of the diagnostic system for BSE and scrapie in Germany. All suspect cases found by rapid testing are reinvestigated and clarified by the National reference laboratory for these diseases which is part of the recently founded Institute of Novel and Emerging Infectious Diseases at the Federal Research Centre for Virus Diseases of Animals located on the isle of Riems. Until the end of 2001 130 BSE cases were confirmed out of 230 submissions.     

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。     

Excretion of BSE and scrapie prions in stools from murine models

Faeces from infected animals have been suggested as a potential source of contamination and transmission of prion diseases in the environment. This work describes the development of a procedure for the detection of PrP(res) in stools which is based on a detergent-based extraction and immunoprecipitation (IP). The procedure was evaluated by analyzing TSE-spiked sheep and mice faeces, and proved to be specific for PrP(res) with sensitivities of 5-10mug of infected brain tissue. In order to analyze the shedding of prions, we studied stools from orally inoculated mice over 4-days post-inoculation and also stools from terminally sick scrapie-infected mice. PrP(res) was only detected in stools shortly after the oral ingestion of TSE agents. The procedure described could be a useful tool for studying the excretion of prions and for evaluating potential environmental contamination by prions.     

Epidemiologic aspects of scrapie and BSE including an analysis of risk factors

After a discussion of the different hypotheses about the causative agent of prion diseases, various aspects of the two most important animal prion diseases, i.e. BSE and scrapie are described. This thesis focuses on the search for a preclinical diagnosis. A major breakthrough was the discovery of a new technique for detecting the disease-associated protein in tonsillar biopsies from scrapie-infected sheep long before clinical signs appeared. Another essential part of the studies described in this thesis concerned a risk analysis for BSE in a country like the Netherlands. Major risk factors were assessed, including an assessment of the efficacy of Dutch rendering procedures in the inactivation of the agents of scrapie and BSE.     

Geographic distribution of BSE in Switzerland

Visual evaluation of the occurrence of BAB (born after ban) cases pointed towards spatial clustering. Therefore a statistical analysis of spatial case clustering was conducted using GIS technology. In the initial analysis, all 376 cases (135 BAB, 241 BBB/born before ban) diagnosed through mid of March 2001 were investigated using the spatial scan statistic. Two clusters of BBB cases were identified in the western part of Switzerland, and two clusters of BAB cases in the eastern part. Epidemiological investigations performed within the BAB clusters showed an increased pig density in these cluster regions. Pig density is considered an indicator for the probability of contamination of cattle feed with feed containing meat-and-bone meal that is intended for other species (cross-contamination). In an updated cluster analysis including all cases reported until end of June 2002 (data set B), clusters were identified in the same regions as previously. It was shown that the BAB clusters occurred in different time periods. The small scale differences in cluster size and location are discussed, with an emphasis on the implications for following epidemiological investigations.     

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.     

Isolation and purification of a Thy-1-like glycoprotein from sheep brain and its distribution in ovine tissues

Standard methods for the purification of Thy-1 were applied to sheep brain to purify a sheep brain membrane glycoprotein (SBMG). On 12 per cent sodium dodecylsulphate polyacrylamide gels this glycoprotein was shown to be a doublet with apparent molecular weights 24,000 and 25,000. By a number of physicochemical criteria SBMG was shown to have properties similar to those previously reported for Thy-1 isolated from other species. Immunological investigations, however, revealed that SBMG did not react with rabbit anti-rat Thy-1 and rabbit anti-SBMG did not recognise rat or chicken brain. By fluorescent antibody techniques the tissue distribution of SBMG appeared to be similar to that of Thy-1 in other species. A small population of peripheral blood and intestinal lymph lymphocytes were stained with anti-SBMG. These results suggest that sheep have an immunologically distinct Thy-1 homologue.     

Incubation of ovine scrapie with environmental matrix results in biological and biochemical changes of PrPSc over time

Ovine scrapie can be transmitted via environmental reservoirs. A pool of ovine scrapie isolates were incubated on soil for one day or thirteen months and eluted prion was used to challenge tg338 mice transgenic for ovine PrP. After one-day incubation on soil, two PrP^Sc phenotypes were present: G₃₃₈ or Apl₃₃₈ii. Thirteen months later some divergent PrP^Sc phenotypes were seen: a mixture of Apl₃₃₈ii with either G₃₃₈ or P_338, and a completely novel PrP^Sc deposition, designated Cag₃₃₈. The data show that prolonged ageing of scrapie prions within an environmental matrix may result in changes in the dominant PrP^Sc biological/biochemical properties.     

Age, scrapie status, PrP genotype and follicular dendritic cells in ovine ileal Peyer's patches

Follicular dendritic cells (FDCs) residing within ileal Peyer's patches (PPs) are of crucial relevance for sheep scrapie early pathogenesis and subsequent scrapie prion neuroinvasion. In this study, ileal PP follicles were significantly more numerous in lambs than in adult Sarda breed sheep, with significant differences being also found in lymphoid follicle area, perimeter and FDC density. Furthermore, PrPd deposition within ileal PPs and host's PrP genotype did not significantly influence these parameters. We conclude that age significantly affects FDC density in ileal PPs from Sarda breed ovines, independently from host's scrapie status and PrP genotype.     

Classical natural ovine scrapie prions detected in practical volumes of blood by lamb and transgenic mouse bioassays

Scrapie is diagnosed antemortem in sheep by detecting misfolded isoforms of prion protein (PrP^Sc) in lymphoid follicles of the rectal mucosa and nictitating membranes. Assay sensitivity is limited if (a) the biopsy is collected early during disease development, (b) an insufficient number of follicles is collected, or (c) peripheral accumulation of PrP^Sc is reduced or delayed. A blood test would be convenient for mass live animal scrapie testing. Currently approved techniques, however, have their own detection limits. Novel detection methods may soon offer a non-animal-based, rapid platform with detection sensitivities that rival the prion bioassay. In anticipation, we sought to determine if diseased animals could be routinely identified with a bioassay using B lymphocytes isolated from blood sample volumes commonly collected for diagnostic purposes in small ruminants. Scrapie transmission was detected in five of six recipient lambs intravenously transfused with B lymphocytes isolated from 5~10 mL of blood from a naturally scrapie-infected sheep. Additionally, scrapie transmission was observed in 18 ovinized transgenic Tg338 mice intracerebrally inoculated with B lymphocytes isolated from 5~10 mL of blood from two naturally scrapie-infected sheep. Based on our findings, we anticipate that these blood sample volumes should be of diagnostic value.     

Development of Purkinje cells in the ovine brain

Purkinje cells are involved in many vital functions within the body. Twenty ovine fetuses ranging from 2 to 5 months of gestation, two lambs in the first week after birth and three adult sheep were studied. Sections of the cerebellum were stained with haematoxylin and eosin, cresyl violet and Klüver-Barrera. This study indicates that Purkinje cells began to appear after the 15(th) week of gestation. There were varying degrees of development of Purkinje cells in different zones of the cerebellum. Our findings in sheep fetuses suggest that the maturation of Purkinje cells starts in the caudal regions of the cerebellum and that the process begins in the vermis before it does in the cerebellar hemispheres. The alignment of Purkinje cells was found to be very regular in the caudal regions of the cerebellum. A partial absence of Purkinje cells in the rostral regions of the cerebellum was observed in both sheep fetuses and adult sheep. In the first post-natal week, some ectopic Purkinje cells were found in the white matter of the cerebellum.