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In the second phase interim published results reported BSE in three of four cattle dosed with 1 g IP range: After publication of the interim findings, two further cases of BSE were diagnosed in cattle in the second phase, one dosed with mg and the other with 10 mg.
For completeness of the data from the second phase the times from exposure to onset of the different clinical stages and cull for all BSE-positive cases are given in Table 1 and for all other cattle where BSE was excluded by postmortem tests in Table 2.
Neuropathological examination confirmed a vacuolar profile in the brain of the case dosed with 10 mg consistent with that reported previously in the study and with that of naturally affected cattle . The animal dosed with mg and culled with spastic syndrome did not present with vacuolar changes in the brain but in both cases the diagnosis of BSE was confirmed by detection of PrPd immunohistochemically and PrPres on WB.
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The clinical duration in confirmed BSE cases time from onset of possible signs to cull, excluding those animals that were culled prior to displaying definite signs ranged from 4 months CM dosed with 1 g to 48 months CM dosed with 1 mg.
It is for this reason that throughout the study IP has fbi iasp writing assessments defined on the basis of onset of definite signs.
No novel polymorphisms were detected in the ORF regions of the 86 tested cattle. Variations in the number of N-terminal octapeptide repeats among PrP gene sequences were reported previously . A summary of the results is given in Table 3.
The findings were consistent with those reported previously . In other words, the main factor that determined how rapidly orally dosed cattle succumbed to BSE was the dose of inoculum, and the genotype did not appear to have a significant influence.
This may be due to sample size in the current study: Using the individual IP data for each confirmed BSE case Table 1 and  for phase 1 of the studythe probability of infection given the dose S d could be expressed as: This ID50 estimate is equivalent to 0.
As it has been shown that one mouse i. ID50 , it can be extrapolated that one cattle oral ID50 equals ID50, previously estimated at Preliminary findings from the original study contributed to quantitative risk assessment of the exposure of humans to consumption of infected bovine products .
An estimate of human ID50 assumed the worst case of a cattle to human species barrier of a factor of one, giving the range of human oral ID50s in 1 g of brain from a clinically affected cow as approximately 0.
Data from the previously published interim results revised this estimate to 1. Although the reduced ID50 based on the present results would increase estimates of the exposure of humans in terms of bovine oral ID50s, the effect would be comparatively small relative to the uncertainty in such risk assessments.
Nevertheless, with decline of the BSE epidemic and the potential for relaxation of certain controls, the revised estimate of human oral ID50 is available to revisit risk assessments.
The present data do not affect the previous approximation that single doses in the range from mg to 1 g of the brainstem homogenate used correspond to the range of mean IPs of cattle through the BSE epidemic .
The observation that a relatively small, single exposure less than 1 g of high titre brain can result in infection reinforces the importance of preventing crosscontamination during feed ingredient storage and feed production.
This proved to be problematical in feed mills producing ruminant and non-ruminant feedstuffs as is evident from the incomplete effect of the initial statutory control on the feeding of meat and bone meal to ruminants introduced in the UK in The low dose phenomenon, together with the persistent viability of the BSE agent, has required the removal of specific high risk tissues from cattle at slaughter and the total ban on the use of mammalian meat and bone meal for use in farmed livestock .
Conclusions The present results concur with the interim findings of this study, that the oral exposure of cattle to BSE brain homogenate produced dose dependent effects on IP and attack rate such that in general the higher the dose the shorter the IPs and the greater the attack rate.
In all cases the induced disease closely resembled the pathology of the natural disease. This is in keeping with the analysis of the pathology in orally dosed cattle from another study  and reinforces the validity of the oral exposure model for the study of classical BSE in the natural host.Botulinum Toxin in Painful Diseases Pain and Headache Vol.
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