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Transmissions to mice were set up from six typical sporadic cases of CJD (spCJD) and three cases of vCJD. All were homozygous for methionine at codon 129 of the ‘prion protein’ (PrP) gene, and none carried PrP gene mutations associated with familial disease. The spCJD cases included two dairy farmers (aged 61 and 64 years) who had had BSE in their herds and had therefore been potentially exposed to BSE-infected cattle or contaminated animal feed11; two ‘contemporary’ cases (aged 55 and 57 years) with no known occupational exposure to BSE; and two ‘historical’ cases (aged 57 and 82 years) who had died in 1981 and 1983, before the onset of the BSE outbreak. All of these spCJD cases were characterized by widespread spongiform vacuolation in the brain with few or no amyloid plaques. The vCJD cases (aged 29, 30 and 31 years) had clinical and neuropathological characteristics that were atypical for CJD9. The main distinguishing neuropathological features in these and other vCJD cases are an extensive deposition of PrP amyloid in the brain as large ‘florid’ plaques and a prominent involvement of the cerebellum.

Panels of three inbred mouse strains (RIII, C57BL and VM) and one cross (C57BL × VM) were challenged with CJD brain homogenates. Previous transmissions, using the same protocol, of BSE from eight unrelated cattle (Fig. 1b) and TSEs from three domestic cats (Fig. 1c), a greater kudu and a nyala (two exotic ruminants) have given a remarkably uniform pattern of incubation periods in these mice5,6,8. The shortest incubation periods were seen in RIII mice, with means ranging from 302 to 335 days for transmissions from frozen brain samples. These isolates also produced strikingly similar patterns of vacuolar degeneration in the brains of infected mice, as represented by the ‘lesion profile’5,6 (Fig. 2b, c). The lesion profile is a well-established semiquantitative method of measuring the targeting of vacuolation to different brain regions, and reliably discriminates between TSE strains in mice2. In addition, the disease characteristics in mice injected with brain from two sheep, a goat and a pig that had been experimentally infected with BSE were very similar to those seen in direct BSE transmissions from cattle6,7.

Figure 1: Incubation times in mice with spongiform encephalopathies.
figure 1

Incubation periods in RIII, C57BL, VM and C57BL × VM mice in transmissions of: a, natural scrapie from six sheep; b, BSE from eight cattle; c, FSE from two cats; and d, three cases of vCJD. C57BL × VM mice were not included in the first, third and fourth BSE transmission; missing symbols elsewhere indicate that no clinical disease was seen in these groups up to the natural lifespan of the mice. The vertical dotted line in d shows the current time after challenge in experiments still in progress. Data are mean ± s.e.m.

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Figure 2: Lesion profiles for mice following transmission of spongiform encephalopathies.
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Lesion profiles are for RIII mice in transmissions of: a, natural scrapie from five of the six sheep from Fig. 1a (n = 3–16 mice per group; no clinical disease was seen in this mouse strain in the sixth transmission); b, BSE from the first four cattle (n = 123, pooled data); c, FSE from two cats (n = 36, pooled data) and TSEs from a greater kudu and a nyala (n = 12 and 11); d, vCJD from three sources (n = 10, 12 and 16) and e, spCJD from two sources, a farmer and a contemporary case (n = 8 and 9). The pooled BSE profile is shown as a dotted line in c–e. As white-matter vacuolation was not a prominent feature in any of these transmissions, only the grey-matter lesion profiles are shown. Vacuolation was scored on a scale of 0–5 in the following scoring areas: 1, dorsal medulla; 2, cerebellar cortex; 3, superior colliculus; 4, hypothalamus; 5, thalamus; 6, hippocampus; 7, septum; 8, retrosplenial and adjacent motor cortex; and 9, cingulate and adjacent motor cortex. Data are mean ± s.e.m.

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The BSE ‘signature’, based on both incubation periods and pathology, has only ever been seen in transmissions from animals suspected or known to have been infected with BSE. It has never been seen throughout an extensive series of transmissions, set up in Edinburgh between 1963 and 1994, of other naturally occurring TSEs (35 sheep and two goats with scrapie, two mink with transmissible mink encephalopathy, and a mule deer with chronic wasting disease). For example, the incubation periods and lesion profiles seen in transmissions from six sheep with scrapie, collected since 1985, are shown in Figs 1a and 2a. Within the same timescale a further two sources of sheep scrapie failed to transmit to mice. In general, natural scrapie transmissions in our own laboratory and elsewhere have given variable results, probably reflecting variation in agent strain amongst the sheep sources12,13.

At the time of writing, the transmissions of vCJD to mice have been in progress for 360 days. The RIII mice injected with all three vCJD sources have developed a progressive clinical disease very similar to BSE, with incubation periods in individual mice ranging from 288 to 351 days. The first signs were nervousness and hypersensitivity, followed by lethargy, weight loss, urinary incontinence and postural abnormalities. Excluding early intercurrent deaths, all RIII mice injected with vCJD have developed disease, with mean incubation periods up to a standard clinical endpoint of 304 ± 4, 306 ± 6 and 310 ± 4 days (±s.e.m.) for three sources (Fig. 1d), within but at the lower end of the range previously seen for BSE and related isolates6 (Fig. 1b, c). Clinical signs are now apparent in some of the C57BL mice, an observation that is also consistent with the BSE pattern (see Fig. 1b). Diagnosis was confirmed for all clinically affected mice by the presence of vacuolar degeneration in the brain and for selected mice in all three experiments by the demonstration of relatively protease-resistant isoforms of PrP (PrPres) in western blots of brain extracts and pathological accumulations of PrP in immunostained brain sections.

The neuropathology in clinically affected RIII mice with vCJD was also similar to that seen in RIII mice with BSE, consisting of a mild-to-moderate grey-matter vacuolation of the hypothalamus, medulla oblongata and septum, and a more severe vacuolation of the cochlear nucleus. Amyloid plaques were not a prominent feature of this pathology. The lesion profiles in RIII mice for the three sources (Fig. 2d) were very similar to each other and also to those in transmissions to RIII mice of BSE (Fig. 2b), TSEs of cats and exotic ruminants (Fig. 2c), and experimental sheep, goat and pig BSE6,7, but differed markedly from those seen in transmissions from sheep with natural scrapie (Fig. 2a). Although results are so far only available for the RIII mouse strain, the striking similarity between vCJD and BSE in these mice, in terms of both incubation periods and pathology, is in itself strong evidence that the same strain of agent is involved in vCJD and BSE.

In contrast to the results with the vCJD sources, no clinical signs of neurological disease have yet been seen in any mice in the six transmissions of spCJD, although they have been in progress for between 600 and 800 days. Figure 3 shows survival curves for RIII mice in these experiments, compared with survival curves in BSE, feline spongiform encephalopathy (FSE) and vCJD transmissions. No significant differences in median survival times were found between RIII groups challenged with the six spCJD sources, or between these groups and saline-injected controls. However, this does not indicate a failure to transmit spCJD, as vacuolar degeneration typical of TSE infection was seen in the brains of some mice dying with intercurrent disease in all six experiments, from about 400 days after challenge. This pathology has so far been seen in 130 of the 156 mice surviving beyond 500 days after injection for which brain was available for histopathological scrutiny. No such changes have been seen in the control mice of any age in this set of experiments, or in mice of the same strains injected with human brain homogenates from patients with amyotrophic lateral sclerosis or laryngeal carcinoma in a previously study14. Western blot and immunohistochemical analyses have demonstrated the accumulation of PrPres in selected brains showing vacuolar pathology, confirming successful transmission of a TSE from all six spCJD sources.

Figure 3: Survival curves for mice following transmission of spongiform encephalopathies.
figure 3

Survival curves are for female RIII mice in transmissions of: a, spCJD from cases with no known occupational exposure to BSE; b, spCJD from two farmers and vCJD from three sources; and c, BSE from the first two cattle sources and FSE from two cats. No distinction is made between mice dying with clinical signs of TSE infection and mice dying with intercurrent disease. Deaths up to 50 days after challenge, most of which were related to injection trauma, are excluded from the analysis.

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A full analysis of the pathology in recipient mice in spCJD transmissions will be presented when these experiments are complete, but already several points can be made. Vacuolar degeneration has been seen in all four mouse strains. This pathology differs in severity between individual mice, but shows a consistent pattern between mouse strains and between spCJD sources. The earliest pathology is seen consistently in the superior colliculus and olfactory tract. In brains showing more widespread vacuolation there is also prominent involvement of the cerebral cortex, thalamus, hypothalamus, caudate nucleus and optic tract, a distribution unlike that seen in BSE transmissions to mice. As an illustration, Fig. 2e shows the lesion profiles for RIII mice killed with intercurrent disease or culled between 500 and 750 days after challenge with two spCJD sources (a farmer and a contemporary case). Although these profiles are not based on animals at the clinical endpoint of the disease, they clearly show a similarity in lesion distribution between the two sources of spCJD, and a difference between these sources and vCJD or BSE, particularly in scoring area 3, the superior colliculus. The results of these transmissions therefore provide no evidence of a link between CJD in dairy farmers and BSE.

A series of transmissions to mice of spCJD and familial human TSEs associated with mutations in the PrP gene have been reported in Japan15. Although different mouse strains were used in the Japanese series, the results for transmissions of spCJD from 129-methionine sources were broadly similar to ours in that transmission was achieved from all sources and mean incubation periods in recipient mice were long (573–863 days)15. Transmissions of the familial TSE Gerstmann–Straussler–Scheinker syndrome (GSS) were achieved from only one-third of the sources tested, but the mean incubation periods in successful transmissions were relatively short (237–517 days)15. Although some of these incubation periods were quite close to our results for vCJD in RIII mice, the pathology in mice with GSS was strikingly different as it included a prominent vacuolation of white-matter tracts16. The Japanese workers also reported the transmission of another human familial TSE, fatal familial insomnia (FFI), with a mean incubation period of 455 days in recipient mice17. The pathology in mice with FFI was indistinguishable from that in mice with spCJD in the Japanese series, apart from there being a more pronounced involvement of the thalamus.

Our results highlight several fundamental features of the TSEs previously established using experimental isolates3,4. The consistency in transmission properties shows that the agent must interact with genetic factors in the host to control the timing and neuropathology of the disease with extraordinary precision. Different strains of agent (spCJD, vCJD) can be isolated from hosts with the same PrP amino-acid sequence (in this case, patients with the 129-methionine genotype) but, conversely, the same strain of agent can be detected in hosts with different PrP sequences (so far the BSE ‘signature’ has been seen in transmissions from eight different species6). This clearly indicates that TSE agents carry some form of information that specifies strain-specific properties, but the molecular basis of this information is still a matter for speculation4.

It has been reported that vCJD can be distinguished from spCJD by the relative prominence of differently glycosylated forms of PrPres and the molecular size of the unglycosylated form18. Samples from dairy farmers with CJD have given glycoform ratios resembling those from other cases of spCJD19. A similarity in glycoform patterns between vCJD and BSE has been presented as evidence of a link between the two18. However, a ‘BSE-like’ glycoform pattern has also been seen for experimental scrapie isolates that are unrelated to BSE20 and for FFI in humans21. Therefore, although the analysis of PrP diversity provides a useful supplement to strain typing in mice, it is premature to draw conclusions concerning causative links between TSEs in different species on the basis of glycoform-ratio analysis alone. A full analysis of glycoform patterns in the present series of transmissions will be reported in due course.

In conclusion, strain typing based on transmission to mice has shown: that vCJD is caused by the same strain of agent that has caused BSE, FSE and TSEs in exotic ruminants; that vCJD is distinguishable from spCJD; and that CJD in two dairy farmers is of the spCJD type and is not linked to the causative agent of BSE. Epidemiological surveillance continues to indicate that vCJD is a new condition occurring almost exclusively in the UK. Our transmission studies, in combination with the surveillance data, provide compelling evidence of a link between BSE and vCJD.

Methods

CJD inocula. The CJD challenge experiments were the first to be undertaken within a new category 3 containment facility at the Neuropathogenesis Unit in Edinburgh, in an environment in which no TSE-infected materials had been handled previously. New dedicated glassware and instruments were autoclaved at 136 °C for 1 h before use. Brain samples for transmission were collected, as far as possible, from areas showing maximum pathology, and stored at −20 °C. Samples were homogenized at 10% (w/v) concentration in sterile physiological saline and stored at −20 °C. Before homogenization of each sample, sterile physiological saline was run through the homogenizer and other glassware and frozen for later inoculation of the appropriate control group. For injection, thawed homogenates were resuspended by being drawn repeatedly through a series of graded needles.

CJD transmissions. Three inbred mouse strains and one cross were challenged: C57BL and RIII (both of the Sinc s7 or Prn-p a genotype), VM (Sinc p7 or Prn-p b genotype), and the F1 cross between C57BL and VM22,23. Groups of approximately 20 mice of each strain were injected by a combination of the intracerebral (20 μl) and intraperitoneal (100 μl) routes under halothane anaesthesia. For each transmission, six mice of each strain were injected with the appropriate saline sample by the same routes. Groups of uninjected control mice were also included. Mice were coded, examined daily throughout their lifespan, and formally scored for signs of neurological disease from 250 days after injection. Mice showing definite signs for two consecutive weeks were killed and incubation periods calculated as the interval between injection and this standard clinical endpoint22. All other mice were maintained to full lifespan, apart from small numbers culled at 700–750 days post-injection, to avoid loss of pathological material.

Histopathological and protein analysis. At post-mortem, a lateral third of each mouse brain was dissected aseptically and frozen at −20 °C for protein analysis and further passage. The remaining two-thirds of each brain was immersion fixed in 10% formol saline for 4 days, treated with 98–100% formic acid for 1 h to inactivate infectivity, and fixed in formol saline for a further 2 days. The brains were trimmed at standard coronal levels and paraffin embedded. Haematoxylin and eosin-stained sections 6 μm thick were prepared, randomly mixed with others from BSE, FSE, sheep scrapie and mouse-passaged scrapie transmissions, and coded for pathological assessment. Vacuolar changes were scored in nine grey-matter and three white-matter areas of brain for the construction of lesion profiles, as described24. PrP in brain sections was immunostained using a polyclonal antibody to mouse PrP, 1A8 (ref. 25), according to a published protocol26. SDS–polyacrylamide gel electrophoresis and western blot analysis of brain tissue were used to confirm the presence of PrPres (ref. 18).

Animal TSE transmissions. The CJD transmissions were compared with transmissions of TSEs from cattle, sheep, domestic cats, a greater kudu and a nyala; the results of some of these animal TSE transmissions have been included in previous publications5,6,8. The design of these experiments was identical to that in the CJD transmissions, except that the source material from one cat, the greater kudu and the nyala was formol-fixed brain. Because transmissions from fixed tissues have resulted in prolonged incubation periods5, probably owing to loss of titre, the incubation period and survival data from these three transmissions are not included in Figs 1 and 3. However, as the pathology in experimentally infected mice is unaffected by the dose of TSE challenge, lesion profile data from the kudu and nyala transmissions are included in Fig. 2.

Statistical analyses. Statistical analyses were performed using the software package Stata, version 5.0. Kaplan–Meier survival curves were plotted and differences in survival between mice inoculated with material from different sources were compared using the log-rank test27. Principal components analysis28 was used to calculate summary measures of lesion profiles and to examine graphically the ‘closeness’ of the lesion profiles from different transmissions. This statistical analysis was in complete agreement with the subjective judgement of ‘closeness’ described in the text and will be documented in detail in a future publication.