Prion diseases: infectious and lethal doses following oral challenge

Michael Baier, Steve Norley, Julia Schultz, Michael Burwinkel, Anja Schwarz and Constanze Riemer

Robert Koch Institute, Project ‘Neurodegenerative Diseases’, Nordufer 20, 13353 Berlin, Germany

Correspondence
Michael Baier
baierm{at}rki.de


   ABSTRACT
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A brain homogenate prepared from a terminally ill hamster infected with scrapie strain 263K was serially diluted and administered orally to groups of hamsters. The undiluted brain homogenate led to clinical scrapie in all animals inoculated. The attack rate decreased with dilutions of the homogenate, and subclinical infections were identified among the healthy survivors at 520 days post-infection by Western blotting. The number of animals succumbing to disease and the combined number of Western blot-positive survivors plus diseased hamsters were used to calculate the LD50 and ID50 of the inoculum. The model system represents an approximation to the transmission of TSEs such as new variant Creutzfeldt–Jakob disease (vCJD) via dietary exposure to the infectious agent and suggests that, due to the rather small difference between the calculated LD50 and ID50, the number of clinical cases will not be vastly exceeded by the number of subclinical carriers of the disease.


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New variant Creutzfeldt–Jakob disease (vCJD) is likely to be initiated by dietary exposure to low doses of the causative agent. Recent calculations concerning the scale of the vCJD epidemic predicted as an upper limit several thousand cases but could not exclude the possibility that many more people, perhaps millions, had been infected but would remain disease-free for life (d'Aignaux et al., 2001; Valleron et al., 2001). Such subclinical carrier patients might pose a considerable risk for contamination of blood products, surgical instruments and tissue transplants. We have studied hamsters infected with the scrapie agent via the oral route to determine the frequency of subclinical infection compared with clinical cases.

A 10 % homogenate prepared from the brain of a terminally ill hamster infected with scrapie strain 263K was serially diluted and administered orally (50 µl) to groups of 25 hamsters. All animals were monitored twice weekly for clinical signs of disease development (Riemer et al., 2000). At 520 days post-infection (p.i.), surviving hamsters were sacrificed and their brain tissue analysed by Western blot using the monoclonal anti-prion protein antibody 3F4 for the presence of the disease-associated, proteinase K-resistant prion protein isoform (PrPSc). Proteinase K digests of 10 % brain homogenates were carried out at 37 °C with 100 µg proteinase K ml-1 for 1 h. The number of animals succumbing to the disease and the combined number of Western blot-positive survivors plus diseased hamsters were used to calculate the LD50 and ID50 of the inoculum using the VacMan program (Spouge, 1992).

The results of the infection experiments are depicted in Table 1. As expected, the undiluted 10 % brain homogenate led to clinical scrapie in all animals inoculated. The attack rate decreased with dilution of the homogenate and subclinical infections were identified among the healthy survivors at 520 days p.i. Because diagnosis of scrapie infection in the healthy survivors was based on Western blot detection of PrPSc, the sensitivity of this test was evaluated. The Western blot analysis routinely detected PrPSc in 10-5–10-6-fold diluted brain homogenates prepared from terminally ill scrapie-infected hamsters (Fig. 1). Thus, it is unlikely that the healthy survivors at 520 days p.i. were not correctly diagnosed by this procedure (Table 1). The calculations using the VacMan program showed that the ID50 (489 ID50 per ml of 10 % brain homogenate) is only slightly higher than the LD50 (332 LD50 per ml of 10 % brain homogenate).


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Table 1. Oral infection of hamsters

 


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Fig. 1. Sensitivity of Western blot detection of proteinase K-resistant PrPSc. The indicated dilutions of a 10 % brain homogenate from a terminally ill scrapie-infected hamster were subjected to proteinase K digestion, blotted and probed with the anti-PrP monoclonal antibody 3F4.

 
Previous studies have shown that subclinical scrapie carrier states can be established following intracerebral infection in resistant species or by using low-dose inoculums (Hill et al., 2000; Race et al., 2001, 2002; Thackray et al., 2002). However, it was not known whether subclinical infection occurs following oral challenge. We determined the frequency of subclinical infection compared with clinical cases in hamsters infected with the scrapie agent via the oral route. Surviving hamsters testing positive for PrPSc were defined as subclinically (and not preclinically) infected because at the time of sacrifice (520 days p.i.; i.e. 239 days after the last animal succumbed to the disease) it was considered highly unlikely that disease would develop within their remaining life-span (Table 1).

To our knowledge, this is the first experimental attempt to calculate the difference between ID50 and LD50 for transmissible spongiform encephalopathies (TSEs) in an animal model system. The model system represents an approximation to the transmission of TSEs such as vCJD via dietary exposure to the infectious agent and suggests that, due to the rather small difference between the calculated LD50 and ID50, the number of clinical cases will not be vastly exceeded by the number of subclinical carriers of the disease. However, an animal model system cannot account for all factors possibly affecting the transmission of vCJD. For example, the susceptibility to vCJD may vary considerably among individuals due to genetic differences and it may well be that different genotypes influence the length of incubation period and the frequency of subclinical infection (Alperovitch et al., 1999; Jackson et al., 2001). In addition, studies in murine scrapie models indicate that numerous as yet unidentified genetic factors can affect TSE incubation times (Lloyd et al., 2001; Manolakou et al., 2001; Stephenson et al., 2000). Interestingly, despite these limitations, our observations are in agreement with calculations based on a large survey in which appendixes and tonsils were tested for the presence of vCJD-associated prion protein accumulation, which suggested that the frequency of infection was approximately of the same order of magnitude as the most recent estimates for the total number of clinical vCJD cases expected in the UK (d'Aignaux et al., 2001; Hilton et al., 2002; Valleron et al., 2001).


   ACKNOWLEDGEMENTS
 
We thank A. Keller, K. Krohn and S. Lichy for expert technical assistance and M. Beekes for advice on the Western blot procedure; This work was funded in part by grant 325-4471-02/45 from the Federal Ministry of Health, Germany.


   REFERENCES
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
Alperovitch, A., Zerr, I., Pocchiari, M. & 7 other authors (1999). Codon 129 prion protein genotype and sporadic Creutzfeldt–Jakob disease. Lancet 353, 1673–1674.[CrossRef][Medline]

d'Aignaux, J. N., Cousens, S. N. & Smith, P. G. (2001). Predictability of the UK variant Creutzfeldt–Jakob disease epidemic. Science 294, 1729–1731.[Abstract/Free Full Text]

Hill, A. F., Joiner, S., Linehan, J., Desbruslais, M., Lantos, P. L. & Collinge, J. (2000). Species-barrier-independent prion replication in apparently resistant species. Proc Natl Acad Sci U S A 97, 10248–10253.[Abstract/Free Full Text]

Hilton, D. A., Ghani, A. C., Conyers, L., Edwards, P., McCardle, L., Penney, M., Ritchie, D. & Ironside, J. W. (2002). Accumulation of prion protein in tonsil and appendix: review of tissue samples. Br Med J 325, 633–634.[Free Full Text]

Jackson, G. S., Beck, J. A., Navarrete, C., Brown, J., Sutton, P. M., Contreras, M. & Collinge, J. (2001). HLA-DQ7 antigen and resistance to variant CJD. Nature 414, 269–270.[CrossRef][Medline]

Lloyd, S. E., Onwuazor, O. N., Beck, J. A., Mallinson, G., Farrall, M., Targonski, P., Collinge, J. & Fisher, E. M. (2001). Identification of multiple quantitative trait loci linked to prion disease incubation period in mice. Proc Natl Acad Sci U S A 98, 6279–6283.[Abstract/Free Full Text]

Manolakou, K., Beaton, J., McConnell, I., Farquar, C., Manson, J., Hastie, N. D., Bruce, M. & Jackson, I. J. (2001). Genetic and environmental factors modify bovine spongiform encephalopathy incubation period in mice. Proc Natl Acad Sci U S A 98, 7402–7407.[Abstract/Free Full Text]

Race, R., Raines, A., Raymond, G. J., Caughey, B. & Chesebro, B. (2001). Long-term subclinical carrier state precedes scrapie replication and adaptation in a resistant species: analogies to bovine spongiform encephalopathy and variant Creutzfeldt–Jakob disease in humans. J Virol 75, 10106–10112.[Abstract/Free Full Text]

Race, R., Meade-White, K., Raines, A., Raymond, G. J., Caughey, B. & Chesebro, B. (2002). Subclinical scrapie infection in a resistant species: persistence, replication, and adaptation of infectivity during four passages. J Infect Dis 186 (Suppl. 2), 166–170.

Riemer, C., Queck, I., Simon, D., Kurth, R. & Baier, M. (2000). Identification of upregulated genes in scrapie-infected brain tissue. J Virol 74, 10245–10248.[Abstract/Free Full Text]

Spouge, J. L. (1992). Statistical analysis of sparse infection data and its implications for retroviral treatment trials in primates. Proc Natl Acad Sci U S A 89, 7581–7585.[Abstract]

Stephenson, D. A., Chiotti, K., Ebeling, C., Groth, D., DeArmond, S. J., Prusiner, S. B. & Carlson, G. A. (2000). Quantitative trait loci affecting prion incubation time in mice. Genomics 69, 47–53.[CrossRef][Medline]

Thackray, A. M., Klein, M. A., Aguzzi, A. & Bujdoso, R. (2002). Chronic subclinical prion disease induced by low-dose inoculum. J Virol 76, 2510–2517.[Abstract/Free Full Text]

Valleron, A. J., Boelle, P. Y., Will, R. & Cesbron, J. Y. (2001). Estimation of epidemic size and incubation time based on age characteristics of vCJD in the United Kingdom. Science 294, 1726–1728.[Abstract/Free Full Text]

Received 10 December 2002; accepted 5 March 2003.



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