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Short Communication |
1 Veterinary Laboratories Agency (VLA-Lasswade), Pentlands Science Park, Bush Loan, Midlothian EH26 0PZ, UK
2 Moredun Research Institute, Pentlands Science Park, Bush Loan, Midlothian EH26 0PZ, UK
Correspondence
Lorenzo González
l.gonzalez{at}vla.defra.gsi.gov.uk
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ABSTRACT |
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). It is the cause of human variant Creutzfeldt–Jakob disease (Bruce et al., 1997; Will et al., 1996), presumably through exposure to contaminated food. The zoonotic nature of BSE prompted titration experiments to determine the minimum infectious dose for cattle and laboratory mice in an attempt to evaluate the risk for humans. Whilst entry of brain tissue into the human food chain is not intentional, titration studies are always performed on this inoculum, as it contains the highest infectivity levels overall, to provide a worst-case scenario. Transmission of BSE to laboratory RIII mice by intracerebral (i.c.) inoculation was first achieved by Fraser et al. (1988), and the first titration studies from individual cattle brains gave titres of around 105.2 mouse (i.c.) ID50 g–1 in RIII and C57BL mice (Fraser et al., 1992). Parallel titration studies of BSE in RIII mice and cattle indicated that the sensitivity of the mouse bioassay [combined i.c. and intraperitoneal (i.p.) routes] was approximately 500-fold less sensitive [titre of 103.3 mouse (i.c.+i.p.) ID50 g–1] than that of the homologous infection [titre of 106.0 cattle (i.c.) ID50 g–1] (Bradley, 2001).
Cattle BSE is not the only cause for concern for human health, particularly since the marked decline in the bovine epidemic. Sheep are the natural host of scrapie but are also susceptible to experimental BSE infection (Foster et al., 1993). Although a comprehensive retrospective study on natural sheep transmissible spongiform encephalopathies (TSEs) has not resulted in the detection of any sheep BSE-like cases (Stack et al., 2006), natural transmission of BSE from experimentally infected dams to their offspring has been documented (Bellworthy et al., 2005). These findings emphasize the need for studies to establish the minimum infectious dose of sheep-passaged BSE, which is the main objective of the study reported here. We also aimed to obtain a measure of the underestimation of the infectivity titre of sheep BSE tissues when titrated across a species barrier in mice.
The inoculum titrated within this comparative experiment was derived from the brainstem of three AHQ/AHQ Romney sheep that developed confirmed TSE after oral dosing with a brainstem homogenate from six cows clinically affected with natural BSE. These three brainstems were pooled and stored as a 10 % (w/v) homogenate (10–1 dilution) in PBS, pH 7.2.
Six groups of five 12-month-old Romney sheep, all of the ARQ/ARQ prion protein (PrP) genotype, were i.c. challenged with 0.5 ml of the above inoculum at dilutions of between 10–3 and 10–8. Five ARR/ARR sheep of the same breed and age were i.c. injected with a 10–3 dilution of the same homogenate, and a further five ARQ/ARQ sheep were inoculated with PBS. Details of the i.c. injection technique for sheep have been published elsewhere (Foster et al., 1993).
Six groups of 20 RIII mice (PRNP genotype s7s7) were inoculated at 6–8 weeks of age with the same inoculum as that used for the sheep, at dilutions ranging from 10–1 to 10–6. Infection was carried out by a combined i.c. (20 µl) and i.p. (100 µl) route using procedures described elsewhere (Fraser et al., 1992); due to a shortage of inoculum, the 10–1 group only received the i.c. injection. Another 20 mice were injected with PBS by the same routes and were used as a negative control.
Sheep and mice were monitored clinically for the development of neurological signs; once these were considered characteristic of TSE, animals were euthanized either by barbiturate overdose (sheep) or by carbon dioxide (mice). Some mice were culled due to intercurrent illness or to old age [approx. 640–650 days post-infection (p.i.)]. Infectious titres for both species were estimated as ID50 using the Spearman–Kärber method (Hamilton et al., 1977). Differences in ID50 titre between sheep and mice and in incubation periods between different dilution groups, in both sheep and mice titration experiments, were assessed for statistical significance using unpaired t-tests (two-tailed P value).
Histopathological (HP) and immunohistochemical (IHC) examinations were performed on representative brain areas from sheep and mice using standard procedures. Tissue sections (5 µm) were stained with haematoxylin and eosin and examined for the presence of spongiform changes. Consecutive sections were subjected to antigen-retrieval procedures, as described elsewhere (González et al., 2002), and to IHC labelling for disease-associated PrP (PrPd) using primary antibodies R145 for sheep tissues (González et al., 2005a) and 1A8 for murine specimens (González et al., 2005b).
All sheep challenged with the 10–3 and 10–4 dilutions succumbed to BSE, confirmed by HP and IHC, as did three out of five sheep challenged with the 10–5 dilution. All of the remaining ARQ/ARQ Romney sheep were still alive at 2200 days post-challenge, i.e. >3 years after the last BSE casualty. Based on these attack-rate figures, the infectious titre of the inoculum was estimated as 105.4 ID50 g–1, with a 95 % confidence interval of 105.0–105.8. Incubation periods ranged from 470 to 562 days (mean 520 days) for the group inoculated with 10–3 dilution, from 483 to 667 days (mean 554 days) for the 10–4 group and from 644 to 828 days (mean 749 days) for the three sheep of the 10–5 group that developed the disease (Fig. 1). Differences in mean incubation periods were not significant between correlative dilutions (Fig. 1), but were significant between the 10–3 and 10–5 dilution groups (P<0.05).
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The phenotype of PrPd accumulation in the brain of the 13 ARQ/ARQ clinically affected sheep has been described in detail previously (González et al., 2005a) and no differences in this respect were observed between the different dilution groups. The PrPd profiles were very similar between ARQ/ARQ and ARR/ARR sheep, although the latter showed more abundant plaque-like accumulations of PrPd (Fig. 2).
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), confirmed by HP and IHC examinations. Thus, the infectious titre of the inoculum was estimated as 105.0 ID50 g–1, with a 95 % confidence interval of 104.8–105.3. Statistical analysis indicated no significant differences between infectious titres obtained in sheep and mice (P=0.21). The mean incubation periods for the 10–1, 10–2 and 10–3 dilution groups were 389, 376 and 423 days, respectively, and 463 days for the 10–4 mice that developed the disease (Fig. 1). Differences in incubation period were statistically significant between the 10–2 and 10–3 dilution groups, and between the 10–3 and 10–4 dilution groups (Fig. 1).
Accumulation of PrPd was most pronounced in the CA2 sector of the hippocampus, the hypothalamus and the deep cerebellar, inferior olivary and vestibular nuclei. Only three types of PrPd deposition were observed: diffuse punctuate in the neuropil, intraneuronal and intramicroglial (Fig. 2). The lesion profile, as determined by the vacuolar degeneration score in nine selected brain areas (Fraser & Dickinson, 1973), was similar in all dilution groups. Spongiform change was most conspicuous in the dorsal nuclei of the medulla and in the hypothalamus, and was least pronounced in the cerebral and cerebellar cortices (Fig. 3).
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, 1992) but also to sheep-passaged BSE, and develop clinical disease with pathological features indistinguishable from those of direct cattle-to-mouse transmission (see Fig. 3). Whether or not an adaptation of the BSE agent to RIII mice has occurred as a result of passage in sheep is difficult to ascertain due to the diversity of inocula used in different studies. Thus, the inoculum used by Fraser et al. (1992) appeared to be more infectious than the one in our study, at least in terms of incubation period (around 40–50 days longer in our study), particularly if we consider that their results were after i.c. injection, whilst ours were after a combined i.c./i.p. injection. This combined route results in shorter incubation periods after high-dose infection (Fraser et al., 1992), explaining why, in our experiment, the 10–1 group (i.c. only) had a slightly longer incubation period than the 10–2 group (i.c.+i.p.). In contrast to these experiments, the cattle inoculum used in the study by Bradley (2001) gave a titre in RIII mice of only 103.3 (i.c.+i.p.) ID50 g–1, which is clearly lower than in our study, whilst Fraser & Foster (1994) reported a titre of 104–105 ID50 g–1, which is similar to ours. Other titrations of BSE-infected sheep brains have recently been carried out in RIII mice with resulting infectious titres of 104 and 103.5 (i.c.+i.p.) ID50 g–1 (S. J. Bellworthy, unpublished results). It would appear, therefore, that the infectiousness of different cattle and sheep brains can be variable, even if these all originate from animals at clinical disease stage.
The commonly accepted notion (Fraser et al., 1992) that the i.p. route makes little contribution at high dilutions enables a certain degree of confidence in the comparison between infectious titres in sheep and mice. The fact that the PrP genotype of the donor sheep (AHQ/AHQ) was different from that of the recipients probably had little relevance in the final sheep titre, as the ARQ/ARQ genotype is considered the most susceptible to BSE, regardless of the origin of the inoculum. Therefore, in this experimental model of sheep-passaged BSE, the effect of the species barrier appeared to be negligible, as the difference of less than half a logarithm between sheep and mouse titres was not statistically significant. This is in contrast to the much higher (approximately 500-fold) difference observed previously between cattle and mice (Bradley, 2001); the precise explanation of these differences is not known.
Adaptation of the BSE agent to sheep as a result of previous passage in sheep does not seem to have occurred. The eight cases of bovine-derived BSE in ARR/ARR sheep described by González et al. (2005a) had an incubation period of 1333±86 days (mean±SEM) after i.c. inoculation of 0.5 ml of a 10–1 brain homogenate with a titre in RIII mice of 102.4 (i.c.+i.p.) ID50 g–1. The four ARR/ARR sheep described in this paper developed clinical BSE after a statistically similar (1469±82 days) incubation period when inoculated with 0.5 ml of a 10–3 (100-fold more diluted than the cattle inoculum) sheep brainstem homogenate with a titre in RIII mice of 105.0 (i.c.+i.p.) ID50 g–1 (more than 100-fold higher than the cattle inoculum). The incubation periods of ARQ/ARQ sheep challenged with those two inocula were also closely similar: 558±11.4 days for 17 sheep inoculated with cattle BSE (González et al., 2005a) and 520±19.4 days for the five sheep inoculated with the 10–3 dilution of the sheep BSE homogenate (P=0.12).
An exact figure of the relative resistance of ARR/ARR sheep compared with ARQ/ARQ animals cannot be provided, as all four ARR/ARR sheep challenged with the 10–3 dilution succumbed to BSE (and the fifth presumably would have done so had it not died from an intercurrent infection) and it is not known whether some would eventually have succumbed to a 10–4 dilution. However, because only three out of five of the ARQ/ARQ sheep inoculated with a 10–5 dilution developed clinical TSE, the maximum susceptibility ratio between ARQ/ARQ and ARR/ARR would be 60 : 1. This ratio might, however, only apply to the i.c. route, as ARR/ARR sheep appear to be much more resistant to infections by other more natural routes such as the oral route, which so far has provided negative results (S. J. Bellworthy, personal communication). The resistance of ARR/ARR sheep to oral challenge is not due to blocked absorption through the gut epithelium (Jeffrey et al., 2006), but to some other aspect of peripheral pathogenesis.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bradley, R. (2001). Bovine spongiform encephalopathy and its relationship to the new variant form of Creutzfeldt–Jakob disease. In Prions. A Challenge for Science and Public Health System. Contributions to Microbiology, vol. 7, pp. 105–144. Edited by H. F. Rabeneau, J. Cinatl & H. W. Basel. Doerr: Karger Publishers.
Bruce, M. E., Will, R. G., Ironside, J. W., McConell, I., Drummond, D., Suttie, A., McCardle, L., Chree, A., Hope, J. & other authors (1997). Transmissions to mice indicate that ‘new variant’ CJD is caused by the BSE agent. Nature 389, 498–501.[CrossRef][Medline]
Foster, J. D., Hope, J. & Fraser, H. (1993). Transmission of bovine spongiform encephalopathy to sheep and goats. Vet Rec 133, 339–341.[Abstract]
Fraser, H. & Dickinson, A. G. (1973). Scrapie in mice: agent-strain differences in the distribution and intensity of grey matter vacuolation. J Comp Pathol 83, 29–40.[CrossRef][Medline]
Fraser, H. & Foster, J. D. (1994). Transmission to mice, sheep and goats and bioassay of bovine tissues. In Transmissible Spongiform Encephalopathies. Proceedings of a Consultation with the Scientific Veterinary Committee of the CEC, pp. 145–149. Edited by R. Bradley & B. Marchant. VI/413/94-EN Brussels, EC.
Fraser, H., McConnell, I., Wells, G. A. H. & Dawson, M. (1988). Transmission of bovine spongiform encephalopathy to mice. Vet Rec 123, 472.[Medline]
Fraser, H., Bruce, M. E., Chree, A., McConnell, I. & Wells, G. A. H. (1992). Transmission of bovine spongiform encephalopathy and scrapie to mice. J Gen Virol 73, 1891–1897.
González, L., Martin, S., Begara-McGorum, I., Hunter, N., Houston, F., Simmons, M. & Jeffrey, M. (2002). Effects of agent strain and host genotype on PrP accumulation in the brain of sheep naturally and experimentally affected with scrapie. J Comp Pathol 126, 17–29.[CrossRef][Medline]
González, L., Martin, S., Houston, F. E., Hunter, N., Reid, H. W., Bellworthy, S. J. & Jeffrey, M. (2005a). Phenotype of disease-associated PrP accumulation in the brain of bovine spongiform encephalopathy experimentally infected sheep. J Gen Virol 86, 827–838.
González, L., Terry, L. & Jeffrey, M. (2005b). Expression of prion protein in the gut of mice infected orally with the 301V murine strain of the bovine spongiform encephalopathy agent. J Comp Pathol 132, 273–282.[CrossRef][Medline]
Hamilton, M. A., Russo, R. C. & Thurston, R. V. (1977). Trimmed Spearman–Karber method for estimating median letal concentrations in toxicity bioassays. Environ Sci Technol 11, 714–719.[CrossRef]
Jeffrey, M., González, L., Espenes, A., Press, C. M., Martin, S., Chaplin, M., Davis, L., Landsverk, T., MacAldowie, C. & other authors (2006). Transportation of prion protein across the intestinal mucosa of scrapie-susceptible and scrapie-resistant sheep. J Pathol 209, 4–14.[CrossRef][Medline]
Stack, M., Jeffrey, M., Gubbins, S., Grimmer, S., González, L., Martin, S., Chaplin, M., Webb, P., Simmons, M. & other authors (2006). Monitoring for bovine spongiform encephalopathy in sheep in Great Britain, 1998–2004. J Gen Virol 87, 2099–2107.
Wells, G. A. H., Scott, A. C., Johnson, C. T., Gunning, R. F., Hancock, R. D., Jeffrey, M., Dawson, M. & Bradley, R. (1987). A novel progressive spongiform encephalopathy in cattle. Vet Rec 121, 419–420.[Medline]
Will, R. G., Ironside, J. W., Zeidler, M., Cousens, S. N., Estibeiro, K., Alperovitch, A., Poser, S., Pocchiari, M., Hofman, A. & Smith, P. G. (1996). A new variant of Creutzfeldt–Jakob disease in the UK. Lancet 347, 921–925.[CrossRef][Medline]
Received 28 July 2006;
accepted 3 October 2006.
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) in each dilution group (x axis). Surviving sheep and mice dying of old age are represented by 
and uninoculated controls by 
. Attack rates (no. confirmed clinical cases out of the no. animals examined, which were the data used to calculate the titres) are also indicated for each dilution group. Statistical differences in incubation periods between dilution groups are indicated: NS, not significant; *, P<0.05; ***, P<0.001.
) constructed as the mean vacuolation (y axis) of mice that developed clinical signs (n=59, all dilution groups; see Fig. 1b