the bovine isolates used in this study have been previously described ; they all exhibited high infectivity levels in bovine prp mice ( 4,7,9 ) . the equivalent of 2 mg of infected bovine brain tissue was injected intracerebrally into tg650 mice . this line of mice overexpresses ( 6-fold ) human prp with methionine at codon 129 ( met ) on a zurich mouse prp null background and has been shown to be fully susceptible to vcjd agent ( 13 ) . the primary transmission of classical bse isolates was inefficient as judged by the absence of clear neurologic signs and by western blot detection of prp in the brain of only 4/25 inoculated mice . the prp banding pattern was essentially similar to that of vcjd ( low molecular mass fragments and predominance of diglycoform species ; figure 1 ) . protease - resistant prion protein ( prp ) in the brains of human prp transgenic mice infected with atypical or zoonotic bovine spongiform encephalopathy ( bse ) agents . a ) representative western blot analysis of prp extracted ( for detailed protocol , see 7 ) from brain homogenates of mice at terminal stage of disease or at end of lifespan after serial transmission of atypical ( l - type and h - type ) or classical bse isolates . the amount of equivalent brain tissue loaded onto the gels was 0.01 mg ( bse ; fr3 isolate , 2nd and 3rd passage ) , 0.3 mg ( l - type and 1st passage of bse ) , and 10 mg ( prp - negative samples ) . b ) ratio of diglycosylated and monoglycosylated prp species in the brains of mice after serial transmission of l - type or bse isolates ( data plotted as means  standard error of the mean ) . primary passage of l - type isolates are represented as triangles ( orange , it ; blue , fr7 ; green , fr10 ; pink , fr11 ) and bse as squares ( light blue , fr3 ; red , ge ) . passages are indicated by unfilled symbols of the same color ( solid line , second passage ; broken line , third passage ) . the ratio was determined after acquisition of prp chemiluminescent signals with a digital imager as previously described ( 7 ) . secondary passages were performed by using prp - negative or prp - positive individual mouse brains . every time brain homogenate from an aged mouse ( > 630 days of age ) was inoculated , transmission was observed in > 80% of the mice , as determined by clinical signs and prp accumulation . the mean survival time was 600 days ( table ; additional data not shown ) . by the third passage , mean survival time approached 500 days , as is usually observed with vcjd cases ( table ; 13 ) . the vcjd - like prp profile was conserved in all 50 positive brains analyzed ( figure 1 ) . first , the 4 l - type isolates induced neurologic disease in almost all mice ; survival times averaged 600700 days ( table ) . the molecular profile was distinct from bse or vcjd ; prominent monoglycosylated prp species resembled those found in cattle ( figure 1 ) . third , we found neither shortening of the survival time on subpassage ( 1 isolate tested , 2 brains ) ( table ; data not shown ) nor change in prp profile ( figure 1 ) . * bse , bovine spongiform encephalopathy ; na , not available ( experiments still ongoing ) . mice with neurologic signs and positive for protease - resistant prion protein by western blotting . days  se of the mean . for mice with negative results , only the range of survival time is given . note that the primary transmission of france ( 3 ) isolate was performed on both hemizygous and homozygous mice . , bse h - type isolates failed to transmit disease to or even infect tg650 mice . none of the inoculated mice had a detectable level of prp in the brain ( 22 analyzed ) . all inoculated mice survived , and none showed a prp signal in the brain ( 15 mice analyzed ) . to further compare the behavior of the 3 bovine prions in tg650 mice , we examined the regional distribution and intensity of prp deposition in the brain ( 14 ) . histoblot analyses ( 3 brains per infection ) were performed on primary ( l - type , h - type ) and secondary passages ( l - type , h - type , bse ) . as shown in figure 2 , l - type and bse agents showed clear differences according to both the aspect and localization of the prp deposits . granular prp deposits were scattered throughout the brain with bse , as has been previously observed with vcjd ( 13 ) . the ventral nuclei of the thalamus , cerebral cortex , oriens layer of the hippocampus , and raphe and tegmentum nuclei of the brain stem were strongly stained . with bse - l , the staining was finer and essentially confined to the habenular , geniculate , and dorsal nuclei of the thalamus ; the lateral hypothalamus ; the lacunosom moleculare layer of the hippocampus ; the superficial gray layer of the superior colliculus ; and the raphe nuclei of the brain stem . finally , prp could not be detected on brain sections from mice inoculated with h - type isolates ( data not shown ) , thus confirming the western blot data . representative histoblots in 4 different anteroposterior sections showing the distribution of disease - specific prp deposits in the brains of tg650 mice infected with bovine spongiform encephalopathy ( bse ) or l - type bse . panels a  d show infection with bse ( second passage of france 3 ) . panels e  h show infection with l - type bse ( first passage of france 7 ) . panels m  p show brain sections of an age - matched , mock - infected mouse , euthanized while healthy at 700 days postinfection , for comparison . note the differing aspect and distribution of prp deposits between brain of mice infected with bse and bse - l ( arrowheads ) . assignment of the positive brain regions has been made according to a mouse brain atlas after digital acquisition . we found that atypical l - type bovine prions can propagate in human prp transgenic mice with no significant transmission barrier . lack of a barrier is supported by the 100% attack rate , the absence of reduction of incubation time on secondary passage , and the conservation of prp electrophoretic profile . in comparison , indeed , 3 passages were necessary to reach a degree of virulence comparable to that of vcjd agent in these mice ( 13 ) , which likely reflects progressive adaptation of the agent to its new host . at variance with the successful transmission of classical bse and l - type agents , h - type agent failed to infect tg650 mice . these mice overexpress human prp and were inoculated intracranially with a low dilution inoculum ( 10% homogenate ) . therefore , this result supports the view that the transmission barrier of bse - h from cattle to humans might be quite robust . it also illustrates the primacy of the strain over prp sequence matching for cross - species transmission of prions ( 15 ) . extrapolation of our data raises the theoretical possibility that the zoonotic risk associated with bse - l prions might be higher than that associated with classical bse , at least for humans carrying the met prp allele . this information underlines the need for more intensive investigations , in particular regarding the tissue tropism of this agent . its ability to colonize lymphoid tissues is a potential , key factor for a successful transmission by peripheral route . this issue is currently being explored in the tg650 mice . although recent data in humanized mice suggested that bse - l agent is likely to be lymphotropic ( 12 ) , preliminary observations in our model suggested that its ability to colonize such tissues is comparatively much lower than that of classical bse agent .
to assess risk for cattle - to - human transmission of prions that cause uncommon forms of bovine spongiform encephalopathy ( bse ) , we inoculated mice expressing human prp met129 with field isolates . unlike classical bse agent , l - type prions appeared to propagate in these mice with no obvious transmission barrier . 
 h - type prions failed to infect the mice .