a retrospective western blot study was performed for all confirmed bse cases diagnosed in france after july 1 , 2001 , as previously described ( 13 ) .
briefly , prpres was extracted using the tesee western blot confirmatory assay ( bio - rad , marnes - la - coquette , france ) according to the manufacturer s instructions . for immunoblotting , antibodies rb1 , 6h4 ( r - biopharm , st .
didier au mont dor , france ) , sha31 from the tesee western blot ( bio - rad ) , and saf84 ( kindly provided by j. grassi , commissariat energie atomique , saclay , france ) were used ; these antibodies recognize the bovine prp sequences 110113 , 156164 , 156163 , and 175180 , respectively .
two western blot assays were conducted on the brainstem samples and used either an antibody against the prp core ( rb1 , 6h4 , or sha31 ) or a c - terminal antibody ( saf84 ) ( 13 ) .
the characteristics of h - type and l - type bse ( 6,13 ) that were sought included 1 ) a higher or lower molecular mass of unglycosylated prpres with core antibodies in h - type and l - type bse compared with c - type bse , 2 ) a lower proportion of diglycosylated prpres in l - type bse , and 3 ) presence of an additional band at 14 kda with saf84 in h - type bse . all cases with features of h - type or l - type bse
were then subjected to further western blot analyses for detailed quantitative analysis of prpres molecular features ( apparent molecular masses and glycoforms proportions ) . among the 645 bse cases confirmed between july 1 , 2001 , and july 1 , 2007 , 7 h - type and 6 l - type isolates were identified ;
these had occurred at a frequency of 03 h - type and 1 l - type case per year , compared with 6219 cases of c - type bse per year ( table ) .
molecular typing of 48 of these 645 samples was not possible because low levels of prpres prevented detailed molecular characterization or because sample amount was insufficient .
all 13 atypical cases were detected in cattle > 8 years of age , from fallen stock ( 9 cases ) or abattoir ( 4 cases ) ; not 1 atypical case was found among the 98 bse cases detected by clinical surveillance during this period .
these 13 atypical cases were diagnosed by the different rapid bse tests routinely used in france : 9 by prionics - check western , lia , or priostrip ( aes , combourg , france ) , 3 by elisa bio - rad , and 1 by idexx herdchek ( idexx laboratories , schiphol - rijk , the netherlands ) . during retrospective interviews ,
the farmer and veterinarian for 6 of these animals reported clinical signs consistent with tse in 3 fallen stock .
this series of 13 cases identified since the beginning of exhaustive active surveillance should be compared with a total of 17.1 million adult cattle tested , of which 3.6 million were > 8 years of age .
in addition to these 13 cases , the first case of atypical ( h - type ) bse was identified in 2000 , during an exhaustive active surveillance program in rendering plants in a limited region of france . * h - type , higher molecular masses of unglycosylated protease - resistant prion protein ( prpres ) ; l - type , lower molecular masses of unglycosylated prpres ; c - type , classic bse ; uc , unclassified . the distribution of bse - infected cattle by birth date ( figure 1 ) shows that 1 or 2 ( h- or l - type cases ) were eventually found positive by rapid tests of the brainstem in each annual birth cohort from 1986 through 1997 , which compares with up to 221 cattle infected with c - type bse born during 19902001 for which bse was diagnosed during 20012007 .
all h - type and l - type bse cases showed similar features ( figure 2 ) regarding the high apparent molecular masses of unglycosylated prpres ( mean difference of 1 kda using 6h4 antibody ) for h - type bse and lower levels of diglycosylated prpres ( mean difference of 35% using 6h4 antibody ) for l - type bse , compared with c - type bse . for l - type bse cases ,
the differences in apparent molecular masses were more obvious for the diglycosylated band ( 1-kda difference using 6h4 , compared with 0.3 kda for the unglycosylated band ) , as previously observed ( 6 ) .
the 14-kda band characteristic of h - type bse was similarly detected in the 8 isolates with saf84 antibody but in none of the other cases classified as l - type or c - type bse .
distribution of bovine spongiform encephalopathy cases identified from july 1 , 2001 , through july 1 , 2007 , by year of cattle birth .
h - type , higher molecular masses of unglycosylated protease - resistant prion protein ( prpres ) ; l - type , lower molecular masses of unglycosylated prpres ; c - type , classic bse .
representative western blot analyses of protease - resistant prion protein ( prpres ) in h - type ( lanes 2 , 3 ) , l - type ( lanes 5 , 6 ) , and c - type ( lanes 1 , 4 , 7 ) cases of bovine spongiform encephalopathy ( bse ) .
bars to the left of the panels indicate the 29.0- , 20.1- , and 14.3-kda marker positions .
h - type , higher molecular masses of unglycosylated prpres ; l - type , lower molecular masses of unglycosylated prpres ; c - type , classic bse .
monoclonal antibodies sha31 and saf84 were used for prpres detection in panels a and b , respectively .
this study involved exhaustive molecular typing of bse cases during a given test period and in a country in which bse testing has been mandatory for all adult slaughtered or fallen cattle .
france tests more animals than any other european country ; 30% of the animals tested in the european union are tested in france .
the estimated frequency of h - type and l - type bse was 0.41 and 0.35 per million adult cattle tested , respectively ( 1.9 and 1.7 in cattle > 8 years of age ) . given the implementation dates of measures to control bse and the birth dates of these bse - infected cattle , foodborne exposure to an infectious agent can not be fully excluded for any of these cattle .
however , the distribution of cattle affected by h- and l - type bse , by year of birth , differs strikingly from that of cattle affected by c - type bse and is consistent with the hypothesis that h - type and l - type bse might represent sporadic prion disorders . in comparison with another sporadic prion disease ,
the annual frequency of sporadic creutzfeldt - jakob disease in humans , which is estimated only by analyses of reported clinically suspect cases , is 12 cases per million .
similar studies in other countries , instead of those free of c - type bse , would be useful .
an alternative hypothesis to foodborne contamination , such as contamination by a scrapie agent , can not be fully excluded , as such contamination has been shown to be a risk factor for scrapie in sheep ( 14 ) .
this study relied on the identification of bse cases by examination of the brainstem only , as derived from our knowledge of c - type bse ( 15 ) .
we can not exclude possible differences in the pathogenesis of atypical bses that might result in underestimation of their frequency , e.g. , involvement of the brainstem at a later stage than with c - type bse .
this possibility is at least suggested by data available for l - type bse , which shows a preferential distribution of abnormal prp in more rostral brain regions ( 4,12 ) .
studies of the pathogenesis of these novel bse forms are thus important for understanding of prion disorders of domestic ruminant species .
 in france , through exhaustive active surveillance , 17.1 million adult cattle were tested for bovine spongiform encephalopathy from july 2001 through july 2007 ; 3.6 million were > 8 years of age . 
 our retrospective western blot study of all 645 confirmed cases found that 7 were h - type and 6 were l - type . 