Robert Koch Institute, Project Neurodegenerative Diseases, Nordufer 20, 13353 Berlin, Germany
Correspondence
Michael Baier
baierm{at}rki.de
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ABSTRACT |
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MAIN TEXT |
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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 10-510-6-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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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
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 10 December 2002;
accepted 5 March 2003.
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