Transmission of the BSE Agent to Mice in the Absence of Detectable Abnormal Prion Protein

See allHide authors and affiliations

Science  17 Jan 1997:
Vol. 275, Issue 5298, pp. 402-404
DOI: 10.1126/science.275.5298.402


The agent responsible for transmissible spongiform encephalopathies (TSEs) is thought to be a malfolded, protease-resistant version (PrPres) of the normal cellular prion protein (PrP). The interspecies transmission of bovine spongiform encephalopathy (BSE) to mice was studied. Although all of the mice injected with homogenate from BSE-infected cattle brain exhibited neurological symptoms and neuronal death, more than 55 percent had no detectable PrPres. During serial passage, PrPres appeared after the agent became adapted to the new host. Thus, PrPres may be involved in species adaptation, but a further unidentified agent may actually transmit BSE.

One of the distinct features of the BSE agent is its high ability to infect other species (1, 2, 3), whereas other TSE agents are easily transmitted only within a species. This species barrier leads to considerable prolongation of the incubation period during interspecies transmission (4). During subsequent experimental passages, TSE agents adapt to the new host: the incubation period shortens and stable pathological properties are acquired (5). According to the prion hypothesis, PrPres (the pathological, protease-resistant isoform of the prion protein) constitutes the infectious agent in TSEs, and replication involves the homotypic interaction between a pathological PrP molecule and the endogenous native protein to produce a conformational conversion to the abnormal isoform. The magnitude of the species barrier would thus be a condition of the extent of congruency between the PrP of the donor species and that of the new host (6). However, this mechanism cannot account for the exceptional ability of the BSE agent to cross the species barrier. This agent has original properties and is suspected to have contaminated humans (2, 7). Thus, we examined BSE transmission and PrPres during primary transmission to mice and in subsequent passages to other mice.

Thirty C57BL/6 mice were inoculated by intracerebral injection of a 25% BSE-infected cattle brain homogenate. After 368 to 719 days, all of the inoculated animals exhibited symptoms of a neurological disease encompassing mainly hindlimb paralysis, tremors, hypersensitivity to stimulation, apathy, and a hunched posture. Biochemical analysis of their brains showed no detectable PrPres accumulation in more than 55% of the mice; these mice were termed PrPres (Figs. 1 and 2) (8). Histological examination revealed neuronal death in all mice, but other classical changes associated with TSEs—that is, neuronal vacuolation and astrocytosis—were limited to the PrPres+ mice (Fig. 3). Neuronal loss was most obvious in the Purkinje cells of the cerebellum, but degenerated neurons were also observed, to a smaller extent, in the CA1 region of the hippocampus. No sign of local inflammation was present. Electron microscopic examination of degenerated cells showed marginalization and clumping of the chromatin, a characteristic of type I apoptosis (Fig. 3E) (9).

Fig. 1.

(A and B) PrPres detection by protein immunoblot (26). In (A), brains of mice at the terminal stage of the disease (4 mg brain equivalent) were analyzed. B1, B10, B6, and B4, first passage from cattle brain; 2PB4-1, second passage from B4 mouse; Control, negative control brain (mouse inoculated with the brain of a healthy cow and killed 800 days after inoculation without clinical signs); Pos, brain pool of mice at terminal stage of experimental scrapie (strain C506M3); Pos/x, dilutions of positive control. In (B), under conditions of maximal sensitivity, the PrPres signal can be detected at a 1→10,000 dilution of the positive control (2.5 μg brain equivalent). Pos, control, and PrPres samples correspond to 25 mg brain equivalent. (C) Similar degradation pattern of PrP with a range of doses of PK in a normal mouse brain and a PrPres brain, showing the absence of PrPres with less resistance to protease than usual in PrPres brains (27).

Fig. 2.

Transmission features of BSE into mice at first, second, and third passage (28). Histograms represent the amount of PrPres (expressed as a percentage of the positive control) in the brains of mice at the terminal stage of neurological disease. Diamonds represent the incubation period for each individual mouse tested for PrPres. The positive control corresponds to a brain pool of mice at the terminal stage of experimental scrapie (strain C506M3). At primary passage, individual mice were scored from B1 to B30 according to their incubation periods. The brains of B2, B3, B26, and B27 could not be analyzed and are not represented. The brains of B1 and B4 were inoculated to a second series of mice called, respectively, 2PB1 and 2PB4. At third passage, the recipient mice were called, respectively, 3PB1 and 3PB4. Second passages were also performed with B6, B10, and B15 and are not shown for the sake of clarity; they were consistent with the passages from B1 and B4.

Fig. 3.

Histological examination of the brains of mice at the terminal stage of disease (29). (A and B) Toluidine blue staining in the cerebellum of a PrPres+ mouse (A) and a PrPres mouse (B). Note the Purkinje cell layer with normal and degenerated cells. Vacuoles in the internal granular layer were seen only in the PrPres+ mouse. These lesions were not seen in aged control mice. Scale bar, 20 μm. (C and D) Immunohistochemistry for GFAP in the thalamus of a PrPres+ mouse (C) and a PrPres mouse (D). The dark staining of protoplasmic astrocytes and the presence of vacuoles were seen only in the PrPres+ mouse. Scale bar, 20 μm. (E) Electron microscopic examination of an apoptotic Purkinje cell in the cerebellum of a PrPres mouse (30). Note the clumping and marginalization of the chromatin, as well as the normal aspect of the nuclear membrane (arrows) and cytoplasmic organelles (arrowheads show the Golgi apparatus and mitochondria). Scale bar, 0.5 μm.

The PrPres mice were infected with a TSE agent because they could transmit a disease exhibiting the classical features of TSE, that is, PrPres accumulation and spongiform lesions (Fig. 2). The brains of PrPres+ mice (for example, B1) and PrPres mice (for example, B4) were used to inoculate a second series of mice. Most of the mice inoculated with PrPres brains developed a classical TSE, but a few presented the PrPres pattern again and the incubation periods remained spread. However, as was observed at primary passage, PrPres+ and PrPres mice had the same range of incubation periods (Fig. 2) (10). Transmission from PrPres+ mice led to an important reduction of incubation time that was very homogeneous (167 ± 2 days, mean ± SEM) with detectable PrPres in all mice (Fig. 2).

A third passage was performed with one mouse from the B1 lineage and two mice from the B4 lineage, only one of which had detectable PrPres (Fig. 2). After inoculation with the PrPres brain, incubation periods were shortened and less variable and all but one of the mice had detectable PrPres at the terminal stage of disease. Transmission from PrPres+ mice gave very similar incubation periods, whether originally inoculated with brain homogenate from the PrPres or PrPres+ lineages. Finally, as a result of this third passage, the PrPres pattern had almost disappeared (Fig. 2). Thus, the PrPres+ pattern had a selective advantage and was associated with the short and homogeneous incubation periods. Therefore, PrPres could be associated with the adaptation of the agent to its new host.

Because we were able to transmit a TSE agent without detectable PrPres upon three passages, infectivity and PrPres can be dissociated [see also (11)]. The similarity of the clinical signs in PrPres and PrPres+ mice suggests that neuronal death was the major determinant of central nervous system function impairment. However, the presence of spongiform lesions and overt gliosis was directly linked to that of PrPres (12). The role of PrPres in the pathogenesis of cerebral damage has been shown in vitro (13), as has the requirement for normal PrP in the development of disease and pathological lesions (14, 15). Thus, PrPres is clearly involved in the pathogenic process of TSEs. However, it may not be the transmissible component of the infectious agent.

This concept is supported by the multiplicity of TSE strains. For example, more than eight different strains can replicate in syngeneic C57BL/6 mice but exhibit specific properties (incubation period, distribution of the lesions, and biochemical features) even though the PrP of the host is the same (16, 17). Some strains are even able to retain their specific properties upon transmission to different hosts with different PrP molecules (1, 16), whereas others undergo phenotypic changes when passaged in a single host (18). Finally, when mice lacking PrP were inoculated with either the Chandler scrapie strain or the mouse-adapted Fukuoka-1 strain of Creutzfeldt-Jakob disease, they did not develop clinical disease, but several brains contained a transmissible agent 20 weeks after inoculation (14, 19).

Because we could transmit a TSE without detectable cerebral PrPres accumulation in the case of interspecies transmission of the BSE agent, the hypothesized existence of an infectious agent in addition to PrPres becomes more likely; in view of the complexity of TSE strain properties, this agent may be a nucleic acid. Moreover, our results suggest a pathogenic mechanism that may account for the peculiar efficacy of the BSE agent in crossing the species barrier. The BSE agent is virulent enough to replicate in the new host without PrPres accumulation. Hence, it is not eliminated, and during replication the agent may acquire the capacity to convert the new host PrP into PrPres. As a result of this adaptation, the transmissible agent would be tightly associated with PrPres, which would confer enhanced virulence and induce the development of classical spongiform lesions.


  1. 1.
  2. 2.
  3. 3.
  4. 4.
  5. 5.
  6. 6.
  7. 7.
  8. 8.
  9. 9.
  10. 10.
  11. 11.
  12. 12.
  13. 13.
  14. 14.
  15. 15.
  16. 16.
  17. 17.
  18. 18.
  19. 19.
  20. 20.
  21. 21.
  22. 22.
  23. 23.
  24. 24.
  25. 25.
  26. 26.
  27. 27.
  28. 28.
  29. 29.
  30. 30.
  31. 31.
View Abstract

Navigate This Article