To purify and identify the infectious agent.
To characterise the PrP genes of domestic species of animals and identify mutations in the human gene that predispose to SE and affect deposition of PrPsc.
To investigate the production of cytokines, prostaglandin and lipocortin during the pathogenesis of scrapie in mice.
To develop transgenic and in vitro models for scrapie, BSE and CID.
1. A highly sensitive method to determine quantitatively the disease-specific amyloid caused by an infection of hamsters with scrapie has been developed and used to study the entrance of the agent from the periphery into the CNS after oral and parenteral infections. The study showed there are several routes into the CNS. One prominent route is via nerves entering the spinal cord in the thoracic section of the cord from where agent replication and amyloid formation move upward towards the brain. This study is continuing in co-operation with the section of histopathology at the Institute for Animal Health at Edinburgh.
2. Building upon work conducted in previous years, conditions for the use of anti-PrP antisera and antibodies in quantitative and qualitative procedures have been deduced and enhanced. Various polyclonal antisera have been raised in chickens and rabbits, primarily using recombinant PrP antigens.
Chicken anti-PrP immunoglobulins have been purified from the egg-yolk. Optimisation of their reactivity has been achieved by utilising antigen affinity chromatography thereby increasing the signal to background noise ratio in procedures such as ELISA and western blotting. The use of recombinant cow and sheep proteins has optimised the reactivity of the antisera for these species PrP. By optimising their efficacy these antisera have proved to be of value in distinguishing PrP5 and PrPsc in small samples of peripheral tissues (spleen, lymph nodes) taken from many experimentally and naturally scrapie-infected sheep. A parallel study of the peripheral tissues of BSE-infected cows has revealed that PrPsc was not detectable. Further studies of PrPSC in BSE-infected cattle tissues will be possible at the greatest possible sensitivity through the application of these immunological reagents.
For sensitive ELISA tests based on antigen competition the use of paired antisera with complementary but not cross-neutralising activities is required. A study using the bacterial recombinant PrP proteins as the ELISA competition targed has determined that such complementary pairs exist within the repertoire of the anti-(recombinant)-PrP polyclonal and monoclonal reagents that has been created. This advancement will allow the development of ELISA tests of the greatest sensitivity for the detection of PrPsc in post-mortem and potentially pre-mortem samples from both sheep and cattle.
3. Polymorphisms in the PrP gene have been studied by analysis of restriction fragments produced by the following restriction enzymes:
A1uI, ApaI, Bam HI, BdIII, EcoRI, EcoRV, HaeIII, HindIII, Hinf1, KpnI, MspI, PstI, SacI, SstI and Tag1 (the enzymes used for cattle are underlined).
In cattle a polymorphism involving the insertion/deletion of a repeated element was shown by PCR and confirmed by sequencing. It is due to the insertion/deletion of a 24 bp sequence to produce two alleles: allele A (373 base pairs) and allele B (349 bp). The frequency of allele A is 94.8% whereas that of allele B is 5.2% within Belgian Blue cattle.
Insertion-type polymorphisms in the canine PrP gene have been searched for by electrophoretic analysis of PCR-amplified genomic fragments and point mutations were looked for by sequencing. No polymorphism could be found among 24 dogs studied.
A variation was observed after PCR amplification in the region of the 24 bp repeats in the PrP gene of cats.
4. We have previously shown that the microglial activation in scrapie shows many features of a restricted form of inflammatory response - there is microglial upregulation of leukocyte antigens with kinown functions in recruitment, phagocytosis (CR3), leukocyte activation (LCA) and antigen prensentation (MHC II). There is also a specific recruitment of monocytes, but not neutrophils or lymphocytes, into affected areas of the brain. Immunocytochemical studies of terminally-affected mice of two scrapie models have demonstrated a marked induction of interleuking-1beta, tumour necrosis factor alpha, prostaglandin (PG) E2, PGF2alpha and liporortin immunoreactivity in affected areas of the brain.
We have now performed time course studies to investigate the relationships between the onset of PrP deposition, glial activation, cytokine immunoreactivity, vacuolation and neuronal apoptosis in the hippocampus and thalamus of the 301V/VM murine scrapie model. The first, focal, PrPSC deposition was present after 30 days of the 115-120 day incubation period; PrPSC immunoreactivity increased in intensity and distribution thereafter. Increased GFAP and F4/80 immunoreactivity, indicating activation of astrocytes and microglia, was evident from 60 days and occurred in areas showing PrPSC deposition. At this time, staining for IL-1b and TNFa was detected in perivascular macrophages, and for PGE2 in astrocytes. Glial cytokine and lipocortin immunoreactivity was detected after 90 days, in the absence of clinical signs. The disease-induced cytokine, PGE2 and lipocortin immunoreactivity occurred only in those brain areas showing PrPSC deposition, activation of astrocytes and microglia. Vacuolation, also confined to areas of PrPSC was evident from 90 days and a major wave of neuronal apoptosis in the CA1 pyramidal layer occurred around 105 days after inoculation. We therefore propose that the infection of neurons by the scrapie agent that leads to PrPSC accumulation also provides a stimulus for glial cytokine production early in the course of the disease. Glial cytokine production would act as a further signal for glial activation. These glial cytokines may also contribute to the development of the other pathological lesions in scrapie. However, whether these cytokines in turn affect PrP metabolism and the neuronal response to infection remains to be determined.
5. As a first step in the investigation of the role of the bovine PrP gene in the pathogenesis and transmission of BSE, the gene has been identified and isolated from a bovine genomic library using a PrP cDNA as a probe. Comparison of the sequence of the bovine PrP gene to those of other species reveals that it is most closely related to the ovine PrP gene. The bovine gene has 3 exons (53,98 and 600 bp) and 2 introns (2,4 and -12 kb). Exon 3 of the bovine PrP gene, like the ovine gene, has a long 3' untranslated region of about 3.8 kb.
Several lines of transgenic mice carrying various numbers of the 3' coding part of exon 3 of the bovine PrP gene linked to the mouse methalliothionein promoter have been shown to express bovine PrP RNA in the brain and / or liver, but they did not make detectable levels of bovine PrP protein. Despite the lack of bovine PrP two of the high copy number lines were inoculated with bovine BSE, but as expected did not show any evidence for reduction in the species barrier.
The construction of several new lines of transgenic mice carrying the bovine PrP gene are underway. The fragment containing the complete exon 3 has been truncated upstream the ORF in order to place the translational initiation codon in an optimal context for efficient translation. The modified exon fragment has been sequenced in the modified 5' region. A second fragment of 800 bp containing only the ORF of the PrP bovine gene has been cloned by PCR and then sequenced. Two promoters have been used in the construction of the transgenic mice; the same methallothionine promoter used previously and the glial fibrillar acid protein (GFAP) gene promoter, which is active in astrocytes.
6. Another goal of the programme has been the investigation of the role of the immune system in the peripheral replication of the TSE agent. A high proportion of SCID mice were shown to be uninfectable with the scrapie agent via the peripheral route. SCID mice, immunologically reconstituted SCID mice, and CD17 were intraperitoneally inoculated with C506M3 scrapie strain. 33% of SCID mice were infected, whereas 97% of reconstituted SCID mice and 100% of CB17 mice developed the disease. PrPSCwas detectable in the brain of all the diseased animals. This indicates the important role that the immune system plays in the peripheral replication of the TSE agent.