experimental groups of 20 ( 4- to 6-week old ) c57bl/6 female mice ( charles river , l'arbresle , france ) were injected intracerebrally with 20 l of 10% ( weight / volume ) homogenates per mouse prepared from brain stem samples of 3 cattle tse isolates .
two of the isolates were characterized , as previously described ( 7 ) , by a higher molecular mass of unglycosylated prp ( h - type isolates ) and labeling with p4 monoclonal antibody ( table ) .
mice were housed and cared for in an appropriate biohazard prevention area ( a3 ) according to european ( directive 86/609/eec ) and french ethical committee ( decree 87848 ) guidelines .
mice were checked at least weekly for neurologic clinical signs and were killed when they exhibited signs of distress or confirmed evolution of clinical signs .
the whole brain of every second mouse was frozen and stored at 80c before western blot analysis .
frozen mouse brain tissues and fixed brain tissues were examined by western blot analysis and immunohistochemical tests as previously described ( 12,13 ) .
prp extracted from half of whole brain was detected with monoclonal antibodies sha31 ( 1:10 from tesee sheep / goat western blot , bio - rad , hercules , ca , usa ) ( 14 ) and 12b2 ( 340 ng / ml ) ( 15 ) . these antibodies are directed against the 144-wedryyre-151 and 88-wgqgg-92 murine amino acid prp sequences , respectively .
antibody 12b2 , which has an n - terminal specificity similar to that of monoclonal antibody p4 , shows poor binding to bse - derived prp , but unlike p4 , binds with high affinity to prion protein from most mammalian species , including mice and cattle .
bound antibodies were detected by using enhanced enzymatic chemiluminescence ( amersham , little chalfont , uk ) or supersignal ( pierce , rockford , il , usa ) and visualized either on film ( biomax , eastman kodak , rochester , ny , usa ) or directly in an image analysis system ( versadoc , bio - rad ) .
molecular masses of prp glycoforms were determined as the average of the center positions of the bands from at least 3 repeated electrophoretic procedures , as measured by comparison with a biotinylated marker ( b2787 , sigma , saint louis , mo , usa ) included on each gel .
immunologic reactivities of antibodies 12b2 and sha31 were compared in western blots run in parallel with the same samples with both antibodies .
after intracerebral injection of cattle brain samples into c57bl/6 mice , disease was observed in mice with the 2 h - type isolates , as well as with the bse sample .
survival periods of mice and results of prp detection among mice analyzed by western blot are shown in the table .
western blot analysis of prp from h - type  infected mouse brains in comparison with bse - infected mice is shown in figure 1 .
this pattern showed higher molecular mass prp glycoforms in mice infected with h - type isolates than in mice infected with a typical bse agent ( 1.1- to 1.5-da difference in the unglycosylated prp ( figure 1a ) .
studies of prp protease cleavage showed that only the prp of mice infected with h - type isolates was recognized by antibody 12b2 ( figure 1b ) .
this finding is in contrast to the result obtained with monoclonal antibody sha31 directed against an epitope in the central region of the protein , which showed that the 12b2 epitope was preserved in h - type  infected mice .
thus , the molecular features of h - type cattle isolates , which are distinct from those of the bse agent , were maintained after development of disease in mice .
western blot analysis of disease - associated prion protein ( prp ) from proteinase k  treated brain homogenates of c57bl/6 mice infected with type h ( lanes 2 and 4 ) or bovine spongiform encephalopathy isolates ( lanes 3 and 5 ) .
prp of mice infected with an experimental scrapie strain ( c506m3 ) ( 6 ) was used as a control ( lane 1 ) .
monoclonal antibodies used for detection of prp were sha31 in panel a and 12b2 in panel b. lane m , molecular mass markers : 39.8 , 29 , 20.1 , and 14.3 kda .
histopathologic analysis showed vacuolar lesions in the thalamus ( figure 2a ) that were absent from the hypothalamus , cochlear nucleus , and superior collicules .
these 3 neuroanatomic sites were severely affected in c57bl/6 mice brain after primary passage of the bse agent as we and others have reported ( 1 ) .
abnormal prp was detected only in amyloid plaques ( figure 2b ) , in contrast to what was reported after bse transmission in c57bl/6 mice ( 1 ) .
a ) characteristic vacuolar lesions in the thalamus ( hematoxylin and eosin stained , scale bar = 60 m ) .
b ) immunohistochemical analysis of prion protein with monoclonal antibody 12b2 ( diluted 1:200 ) shows the absence of granular deposition , but the presence of plaques in the thalamus .
the inset shows that plaques are amyloids since they bind congo red and show birefringence in polarized light ( scale bar = 60 m , scale bar in inset = 16 m ) .
our data show that the recently identified bovine h - type isolates involve an infectious agent that can induce development of a disease across a species barrier , while maintaining the specific associated prp molecular signature .
this evidence in favor of a new bovine prion strain in cattle suggests that bse is not the only transmissible prion disease in cattle .
these cases suggest either the existence of alternative origins of such diseases in cattle or phenotypic changes of prp after infection with the bse agent .
however , based on analysis of molecular features of prion diseases in cattle , this situation is similar to that in humans ( 5 ) , in which different subtypes of sporadic creutzfeldt - jakob disease agents are found .
 we previously reported that cattle were affected by a prion disorder that differed from bovine spongiform encephalopathy ( bse ) by showing distinct molecular features of disease - associated protease - resistant prion protein ( prpres ) . 
 we show that intracerebral injection of such isolates into c57bl/6 mice produces a disease with preservation of prpres molecular features distinct from bse . 