This project will study the early pathogenesis of scrapie and bovine spongiform encephalopathy (BSE) in sheep and mice, with emphasis on the uptake and accumulation of the prion protein (PrP), and infectivity in the gut-associated and peripheral lymphoid tissues and its relevance to neuroinvasion. The transport of PrP across the mucosa of the gut and the interaction of disease-associated PrP with leukocytes, FDCs and nerves will be studied in novel systems such as isolated intestinal loops, surgical micro-infection, B-cell depleted sheep and transgenic mice over-expressing ovine PrP. The consequences of accumulation of PrP in lymphoid tissues for neuroinvasion and the development of a carrier state will be assessed. A better understanding of the early pathogenesis of scrapie and BSE in sheep will contribute the detection of the pre-clinical disease and in developing future control and eradication strategies.
The objectives of the project are to:
1) investigate the role of mucosal transport systems and the involvement of gut-associated lymphoid tissues in the uptake and passage of scrapie and BSE across the intestinal mucosa
2) study key cellular events in peripheral lymphoid tissues related to the establishment of scrapie and BSE infection, and their influence on neuroinvasion, immunomorphology and infectivity
3) evaluate the implications of early peripheral pathogenesis of scrapie and BSE for the carrier state and control strategies.
Progress to Date
In WP1, analysis of material from the gut loop experiments revealed that inoculum was present initially in the lacteals of the villi rather than the dome of the Peyer's patch, suggesting transcytosis of scrapie across non-GALT sites rather than GALT sites of the gut mucosa (Deliverable No. 4). However, this material also demonstrated replication of the scrapie agent 30 days after inoculation but only in susceptible sheep and only at GALT sites (Deliverable No. 5). The results of these studies are in preparation for publication. The limited detection of inoculum in gut loop experiments was investigated further through a series of experiments addressing the pre-digestion of homogenates of scrapie and normal brain using samples of alimentary juices. The Western blot study suggested that the predigestion process removes all normal PrPc and a substantial fraction of PrPres but does not completely digest all of it under conditions of digestion used in this study. These results support the limited detection of PrP by immunohistochemical techniques previously reported. An investigation of gut loops in sheep with resistant PrP genotypes (Milestone No. 8) was performed and examination of this material has shown that both susceptible and resistant PrP genotypes take up the inoculum during the first 2 hours following inoculation (Milestone No. 9). These studies have contributed new information on the transcytosis of scrapie inoculum and PrPd distribution in the gut mucosa of resistant sheep (Deliverable No. 9)
In WP2, the third inoculations of Norwegian white sheep for the investigation of the temporal distribution of PrPd were performed (Milestone No. 26) and the planned necropsies and tissue sample collections were undertaken (Milestone No. 28). The assessment of the influence of PrP genotype on the distribution and accumulation of PrPd, particularly in sheep with less susceptible or resistant PrP genotypes is in progress. A simple and quick biopsy technique has been developed for use in live animals and biopsies collected from sheep with experimental and natural scrapie were used to demonstrate PrPSc using immunohistochemistry, an ELISA test and Western blotting. The morphometric examination of B-cell follicles has shown that follicle size, percentage of T-cells and percentage of proliferating cells were not significantly different in scrapie-infected lambs compared with normal lambs. The investigation of further parameters is in progress.
In WP3, the birth of VRQ/ARR lambs in the spring of 2004 (Milestone No. 31) enabled further experimental infection studies to be performed. Five lambs were experimentally inoculated with scrapie either at the intra-abdominal sites of the rumen wall and spleen or in the prescapular lymph node (Milestone No. 32).
In WP4, the selection and the source of samples to be inoculated into transgenic mice were discussed among the Partners at the third annual co-ordination meeting. It was agreed that tissues from experimental scrapie infected sheep would be transferred from the laboratories of Partner 2 to Partner 4 (Milestone No. 39). Subsequently a transgenic mouse bioassay designed by Partner 4 would be initiated using the frozen distal jejunal lymph node material (Milestone No. 40).
ANIMALS, HUMAN HEALTH AND WELLBEING, GENOMICS
Scientist responsible for the project
Mr JON TEIGE
Ullevaalsveien 72 Box 8146
Norway - NO
Phone: +47 22964500
Fax: +47 224764
||NORWEGIAN SCHOOL OF VETERINARY SCIENCE
||01 December 2001
||1 927 534 €
|Total EC contribution
||1 368 742 €