the animal ethics committee and animal care and use committee of the national institute of animal health approved the study .
five holstein calves 23 months of age were intracerebrally injected with 1 ml of 10% ( w / v ) brain homogenates prepared from the medulla oblongata of bse / jp24 . in our earlier report , experimentally challenged cattle appeared to display clinical signs indicative of bse at 11 months postinoculation ( mpi ) ( 9 ) .
animals were sequentially euthanized before and after the onset of clinical signs ( cattle identification codes 8515 and 496 at 10 and 12 mpi , respectively ) and at the terminal stage of the disease ( cattle identification codes 528 , 1061 , and 5566 at 16 mpi ) .
we provisionally categorized the adrenal gland as nerve tissue because of the presence of chromaffin cells in the medulla of the gland .
western blot analysis for prpres was performed on obex tissue samples as described previously by using anti - prp monoclonal antibody t2 ( 9 ) .
prpres was detectable in all obex samples obtained 10 , 12 , and 16 mpi , suggesting that transmission of l - type bse prions to these animals was successful .
dilution of the protease - treated brain sample and analysis of western blot results showed that the detection threshold for prpres was 1.25 g of brain tissue equivalent ( data not shown ) .
a variety of nerve and lymphoid tissue samples were investigated for accumulation of prpres by western blot analysis by using phosphotungstic acid precipitation , as described previously ( 10 ) ; examples of cattle tissue samples obtained 10 and 16 months mpi ( codes 8515 and 1061 , respectively ) are shown in figure 1 . in cattle at the preclinical stage , prpres was detectable in all tested ganglia and barely detectable in the vagus nerve and vagosympathic trunk . in cattle at the terminal stage ,
prpres was barely detectable in the forelimb nerves ( suprascapular nerve , brachial nerve plexus , median nerve , and radial nerve ) , whereas substantial amounts of prpres were present in other nerve tissues except for facial and hypoglossal nerves ( table ) . a broader nerve tissue distribution of prpres was observed in cattle at 16 mpi than at 10 and 12 mpi .
contrary to what we found in nerve tissues , we detected no prpres from tests performed on lymphoid tissues obtained from any of the 5 cattle studied .
western blot analysis of a protease - resistant form ( prpres ) of a normal cellular prion protein in nerve tissue samples obtained from cattle 10 ( a ) and 16 ( b ) months postinoculation ( cattle identification codes 8515 and 1061 , respectively ) .
the nerve tissues tested are shown above the lanes : 1 , trigeminal ganglia ; 2 , pituitary gland ; 3 , anterior cervical ganglion ; 4 , facial nerve ; 5 , hypoglossal nerve ; 6 , cranial mesenteric ganglia ; 7 , vagus nerve ( cervical part ) ; 8 , stellate ganglia ; 9 , adrenal gland ; 10 , phrenic nerve ; 11 , vagus nerve ( pectoral part ) ; 12 , vagosympathic trunk ( pectoral part ) ; 13 , vagosympathetic trunk ( lumbar part ) ; 14 , accessory nerve ; 15 , suprascapular nerve ; 16 , brachial nerve plexus ; 17 , median nerve ; 18 , radial nerve ; 19 , sciatic nerve ; 20 , tibial nerve , 21 , middle cervical ganglion .
molecular mass standards ( kda ) are indicated on the left of each panel . * prpres , prion protein resistant ; bse , bovine spongiform encephalopathy ; mpi , months postinoculation ; + , positive for prpres ;  , negative for prpres . infectivity of selected nerve tissues ( including the obex , sciatic nerve , adrenal gland , brachial nerve plexus , and vagus nerve ) obtained from cattle euthanized at 10 , 12 , and 16 mpi ( codes 8515 , 498 , and 5566 , respectively ) was analyzed by intracerebral injection into mice transgenic for bovine prion protein , as described previously ( 11 ) . as a negative control , mice were injected with cells from the brainstem of a normal cow .
the presence of prpres in the brains of all mice used in the experiment was determined by western blot analysis .
infectivity was detected in all nerve tissues tested , regardless of the presence of detectable prpres ( figure 2 ) .
inocula from selected tissues  obex ( a ) , sciatic nerve ( b ) , adrenal gland ( c ) , branchial nerve plexus ( d ) , and vagus nerve cervical part ( e)were prepared from cattle euthanized at 10 ( code 8515 , circle ) , 12 ( code 498 , square ) , and 16 ( code 5566 , triangle ) months postinoculation were and inoculated intracerebrally into mice transgenic for bovine prion protein .
ratios above graph indicate number of prion - diseased mice / number of inoculated mice at 500 d postinoculation .
we report accumulation of l - type atypical bse prions in peripheral nerve tissues sampled from intracerebrally challenged cattle .
our study demonstrated that almost all of the peripheral nerve tissues tested became prpres positive in a time - dependent manner , whereas no prpres was detectable in lymphoid tissues , even in cattle with fatal atypical bse .
our results suggest the possibility that , like classical bse prions , l - type bse prions propagated in the central nervous system and were spread centrifugally by nerve pathways ( 11,12 ) . in italy ,
l - type bse prions have been characterized in detail by using cattle challenged intracerebrally .
however , prpres was not detected in their peripheral tissues , including the peripheral nerves ( 13 ) .
the reason for the discrepancy in prpres detection is unclear . in view of the similarities between
the l - type and bse / jp24 prion characteristics ( 9 ) , this discrepancy may result from differences in the methods used for prpres detection .
we detected infectivity in the nerve tissue samples ( including samples from the obex , sciatic nerve , adrenal gland , brachial nerve plexus , and vagus nerve ) obtained 10 , 12 , and 16 mpi .
on the basis of the incubation time of 223  25 ( mean  sd ) days in mice injected with a 1,000-fold dilution of the obex homogenate , infectious titers in peripheral nerve tissues appeared to be 1,000  lower than those estimated in the obex during endpoint titration of infectivity .
our results demonstrate that l - type atypical bse prions can be distributed in the peripheral nerve tissues of intracerebrally challenged cattle .
these findings are useful for understanding l - type bse pathogenesis and accurately assessing the risks associated with this disease .
investigations of prion distribution in cattle that have been orally challenged with l - type bse prions are critical .
 we recently reported the intraspecies transmission of l - type atypical bovine spongiform encephalopathy ( bse ) . to clarify the peripheral pathogenesis of l - type bse 
 , we studied prion distribution in nerve and lymphoid tissues obtained from experimentally challenged cattle . as with classical bse prions , l - type bse prions accumulated in central and peripheral nerve tissues 
 . 
The Study
Conclusions