Volume 2: Science
4. The link between BSE and vCJD
Experimental studies into a BSE/CJD link

4.6 Initial studies in 1988 aimed to test the likely transmissibility of BSE to humans by the inoculation of marmosets, a primate species. Two animals were inoculated intracerebrally and succumbed to disease in early 1992. However, it was known that scrapie was readily transmissible to marmosets (albeit with a shorter incubation period), so the results were not thought to be surprising. Thus, the study did not offer significant insights into the transmissibility of BSE to humans. Once vCJD had been recognised, a host of studies were initiated to investigate a possible link between BSE and vCJD. These studies were reported after March 1996. At that time, evidence for the link between the diseases was only circumstantial, and some doubted its existence. We have examined events occurring after 20 March in order to resolve these doubts and to consider the adequacy of the response to the emergence of vCJD prior to March 1996.

4.7 Strain-typing studies were undertaken in both mice and primates to characterise the pattern of disease in terms of the incubation period and disease pathology of different transmissible spongiform encephalopathies (TSEs). In the study carried out jointly by the Neuropathogenesis Unit (NPU) and CJDSU which began in early 1995, mice were inoculated intracerebrally and intraperitoneally with infected brain homogenate. The disease patterns of BSE, what later became known as vCJD, feline spongiform encephalopathy (FSE) and TSEs of exotic ruminants were shown to be extremely similar, while differing from those of scrapie and sporadic CJD. ¹ Another study in macaques by Lasmezas' group showed that the clinical, neuropathological and molecular features of disease resulting from intracerebral inoculation of BSE brain homogenate were very similar to those observed in vCJD. ² These results provided positive evidence that vCJD and BSE were caused by the same strain of agent.

4.8 Independent confirmation of a link was provided by the western blotting studies of Professor Collinge's group, published in 1996 and 1997. ³ (The western blotting technique is described in Chapter 1). Results showed that in samples of brain digested with proteolytic enzymes and subjected to gel electrophoresis, three bands of protease-resistant prion protein could be visualised using labelled antibody. The size of band depends on the number of sugar molecules bound to the protein molecule by a cellular reaction termed glycosylation. PrP^Sc may have one, two or no sugars bound to the molecule, and these variations lead to the appearance of three bands on the western blot; the fraction with two sugars migrates more slowly than the others during electrophoresis. The pattern of bands is characteristic of the strain of PrP^Sc and the DNA polymorphism at codon 129. There are four main patterns, depending on the relative proportion of each fraction and the conformation of the PrP^Sc molecule. Sporadic CJD was found to be type 1 or type 2, depending on the variant at codon 129. Most iatrogenic CJD cases have a type 3 pattern. Variant CJD and BSE have a type 4 pattern. Samples from cats with FSE and from zoo species affected with TSEs also have the type 4 pattern, consistent with an origin from the BSE strain of PrP^Sc. These data, together with the temporal and geographical association of vCJD and BSE, provide compelling evidence that the two diseases were caused by the same prion strain.

4.9 Recent work has identified additional human PrP^Sc types. Type 4t PrP^Sc has been discovered in lymphoreticular tissue obtained from vCJD patients but not from patients with other prion diseases. ⁴ The pattern of type 4t was found to be consistent, but slightly different from that seen in vCJD brain (type 4). ⁵ Further types (6, 7 and 8) are currently under investigation. ⁶

4.10 These additional types are not, however, universally accepted, since similar analysis by another group could only identify two patterns of prion glycosylation, with vCJD segregating with sporadic CJD. ⁷ Variant CJD could be discriminated on the basis of its glycoform ratio, though the wisdom of doing so was questioned since glycosylation is a co- and post-translational event, and hence likely to be affected by the cell type of the species in which it occurs.

4.11 More recently, important work has shown that the discrepancies between these two studies could be due to the effect of metal ions on the prion protein. ⁸ Both type 1 and type 2 PrP^Sc (ie, sporadic CJD) showed altered glycosylation patterns following the removal of metal ions prior to protease digestion. Rather than producing their distinct patterns, both types gave indistinguishable and common fragment patterns. Similar treatment of PrP^Sc types 3 and 4 (iatrogenic and vCJD respectively) did not alter their characteristic cleavage pattern. These results showed that the respective conformations of type 1 and type 2 PrP^Sc are dependent on the presence of metal ions and that metal ion depletion induces a conformational change in the protein, exposing a new proteolytic cleavage site that is common to both metal-ion-depleted conformers. These findings not only explained the discrepancies between the two studies, but also provided a simple post-translational mechanism that could be involved in conferring strain-specific properties to distinct PrP^Sc conformers.

4.12 Transgenic mice have also provided a powerful tool for the comparison of BSE and vCJD. Replacement of the PrP gene in mice with that from another species effectively abolishes the species barrier, enabling the study of disease in an appropriate experimental model. Mice containing the human PrP gene inoculated with either BSE or vCJD showed similar clinical symptoms and histopathological patterns, which were distinct from sporadic or iatrogenic CJD. ⁹ Western blot analysis of digested brain material again revealed similar glycoform patterns for both BSE and vCJD infected groups. More recent studies by Prusiner have shown that the incubation period and neuropathology of BSE in mice transgenic for the bovine PrP gene are almost exactly the same as those of vCJD in the same breed of mice, though different from scrapie. ¹⁰

4.13 A striking feature of vCJD has been the young age of its victims. The youngest patient recorded to date developed the disease at the age of 12 and the oldest at the age of 55. ¹¹ The average age of onset is about 29 years of age. Observations in patients who have developed CJD following treatment with contaminated growth hormone suggest a range of incubation periods from 5 to 15 years, with an average of 13 years. If the incubation period were similar in vCJD (and there are no data to support this assumption), the average age of infection in vCJD might be around 15 years of age.

4.14 A widely held view is that vCJD was transmitted in beef products in which parts of the animal containing high titres of BSE infection were included: especially brain, spinal cord and dorsal root ganglia. Such tissues might be expected to be found in mechanically recovered meat. It is therefore relevant to consider if processed beef products containing these materials were disproportionately consumed by children and young adults. Our attention was brought to a study in which the age-related consumption of these products was considered in a group of individuals aged 15 years and over. ¹² It was found that the consumption of beef, sausages, meat pies and corned beef was not correlated to the age of the individual. However, in the case of burgers, the consumption fell steeply with age and furthermore was correlated with the age distribution of the first 20 cases of vCJD. Burgers were the only meat product to show a steep decline in consumption with age. The study notes evidence which indicates that among all meat products, homogenates of cow brain have been used commonly to bind ground beef in burgers. ¹³ The use of brain in food was banned in the UK in 1989, and we have received some contrary evidence that brain was seldom included in meat products even before that date. ¹⁴ Nonetheless, the use of MRM in processed food remains a strong candidate as a possible route of infection in young vCJD patients. Other possibilities which remain to be tested and which might account for the age distribution of vCJD patients include:

1. Increased incidence of infections or ulcers of the nasopharynx, such as tonsillitis, or of the lower intestinal tract, such as gastroenteritis, in children compared with adults. There is evidence that TSEs may transmit through broken skin and mucous membranes, such as those that line the gastrointestinal tract.
2. Infection through gum lesions associated with eruption of teeth.
3. Iatrogenic transmission via vaccines prepared in cultures containing bovine constituents.

4.15 With regard to the possibility of genetic susceptibility, the only evidence of this comes from the observation that all vCJD patients (now numbering more than 50) are homozygous for the methionine variant at codon 129 of the PRNP gene. As this variant occurs in 38 per cent of the population, many other factors (including polymorphisms at other genetic loci) must also be involved in susceptibility to this disease. This is discussed further in vol. 8: Variant CJD.