Past, present and future of bovine spongiform encephalopathy in France.

The bovine spongiform encephalopathy (BSE) epidemic has been monitored in France since the end of 1990. The surveillance has been considerably enhanced since 2000, and today every cow aged 2 years or more is tested at the time of slaughter, culling or death. As of 1 May 2002, 613 native cases have been identified, 287 of them by the mandatory reporting system of suspect clinical cases or complementary programs, 213 by active surveillance of fallen stock and 113 by testing at the abattoir. The analysis of reported BSE cases shows a higher number of cases born between 1993 and 1995, which can be linked to a greater exposure at that time and to an increase in surveillance efficiency. When the clinical onset related to overexposure ends, the future trend of the BSE epidemic in France will depend on the efficiency of the control measures implemented since 1996. An indicator of this will be the number of BSE cases born among recent cohorts. Bovine spongiform encephalopathy (BSE) was described first in 1987 in Great Britain (Wells et al. 1987), where a huge epidemic started, and more than 180,000 cases have been detected since then in this country. A risk analysis undertaken in France in 1990, in part because of the amount of meat and bone meal (MBM) imported from Great Britain during the 1980s, led to the conclusion that BSE might have spread to France, and that sporadic cases might be observed (Savey et al. 1991). Epidemiological surveillance was therefore set up at the end of 1990, and control measures were taken to prevent the development of the disease. The trends in the number of BSE cases detected in France must be analyzed in the light of both the detection system and the control measures, as well as their changes over time.

Immunohistochemistry of PrPsc within bovine spongiform encephalopathy brain samples with graded autolysis.

Bovine spongiform encephalopathy (BSE) is a transmissible neurodegenerative disease of cattle. Clinical diagnosis can be confirmed by investigation of both spongiform changes and abnormal prion protein (PrPsc), a marker considered specific for the disease. Tissue autolysis, often unavoidable in routine field cases, is not compatible with histological examination of the brain even though PrPsc is still detectable by immunoblotting. To determine how autolysis might affect accurate diagnosis using PrPsc immunohistochemistry, we studied 50 field samples of BSE brainstem (obex) with various degrees of autolysis. We demonstrated that the antigen-unmasking pretreatments necessary for PrPsc immunohistochemistry were compatible with the preservation of autolyzed brain sections and that PrPsc detection was unaffected by autolysis, even though anatomic markers were sometimes lost. In tissue samples in which anatomic sites were still recognizable, PrPsc accumulation was detected in specific gray matter nuclei. In samples with advanced autolysis, PrPsc deposits were still observed, at least at the cellular level, as an intraneuronal pattern. We found that the sensitivity of PrPsc immunohistochemistry as a diagnostic method for BSE was undiminished even by severe tissue autolysis.

Molecular analysis of the abnormal prion protein during coinfection of mice by bovine spongiform encephalopathy and a scrapie agent.

Molecular features of the proteinase K-resistant prion protein (PrP res) may discriminate among prion strains, and a specific signature could be found during infection by the infectious agent causing bovine spongiform encephalopathy (BSE). To investigate the molecular basis of BSE adaptation and selection, we established a model of coinfection of mice by both BSE and a sheep scrapie strain (C506M3). We now show that the PrP res features in these mice, characterized by glycoform ratios and electrophoretic mobilities, may be undistinguishable from those found in mice infected with scrapie only, including when mice were inoculated by both strains at the same time and by the same intracerebral inoculation route. Western blot analysis using different antibodies against sequences near the putative N-terminal end of PrP res also demonstrated differences in the main proteinase K cleavage sites between mice showing either the BSE or scrapie PrP res profile. These results, which may be linked to higher levels of PrP res associated with infection by scrapie, were similar following a challenge by a higher dose of the BSE agent during coinfection by both strains intracerebrally. Whereas PrP res extraction methods used allowed us to distinguish type 1 and type 2 PrP res, differing, like BSE and scrapie, by their electrophoretic mobilities, in the same brain region of some patients with Creutzfeldt-Jakob disease, analysis of in vitro mixtures of BSE and scrapie brain homogenates did not allow us to distinguish BSE and scrapie PrP res. These results suggest that the BSE agent, the origin of which remains unknown so far but which may have arisen from a sheep scrapie agent, may be hidden by a scrapie strain during attempts to identify it by molecular studies and following transmission of the disease in mice.

Comparison of French natural scrapie isolates with bovine spongiform encephalopathy and experimental scrapie infected sheep.

We compared the glycoform pattern of the abnormal prion protein (PrP(Sc)) detected by immunoblotting in 21 sheep with natural scrapie, from 21 different outbreaks identified in France since 1996, with a bovine spongiform encephalopathy (BSE)-infected sheep. All the natural scrapie isolates had a higher molecular mass of the unglycosylated PrP(Sc) than in BSE-infected sheep. In the latter case, this molecular mass appeared identical to that found in the CH 1641 experimental scrapie strain (type C pattern), whereas in natural scrapie cases it was similar to that found in the SSBP/1 experimental scrapie strains. These results suggest that all French natural scrapie isolates studied so far would belong, as SSBP/1, to the group of scrapie cases with type A electrophoretic pattern.

Similar signature of the prion protein in natural sheep scrapie and bovine spongiform encephalopathy-linked diseases.

It has been suggested that specific molecular features could characterize the protease-resistant prion protein (PrP res) detected in animal species as well as in humans infected by the infectious agent strain that causes bovine spongiform encephalopathy (BSE). Studies of glycoform patterns in such diseases in French cattle and cheetahs, as well as in mice infected by isolates from both species, revealed this characteristic molecular signature. Similar studies of 42 French isolates of natural scrapie, from 21 different flocks in different regions of France, however, showed levels of the three glycoforms comparable to those found in BSE-linked diseases. Moreover, the apparent molecular size of the unglycosylated form was also indistinguishable among all different sheep isolates, as well as isolates from BSE in cattle. Overall results suggest that scrapie cases with features similar to those of BSE could be found more frequently in sheep than previously described.

Immunological characterization of the sheep prion protein expressed as fusion proteins in Escherichia coli.

The prion protein (PrP) from sheep was produced in large quantities of entire protein in Escherichia coli after fusion with a carboxy-terminal hexahistidine sequence. In contrast, amino-terminal fusion with glutathione S-transferase (GST) revealed a high susceptibility toward cleavage of the protein. Both recombinant proteins were recognised, at variable levels, in Western blots using a panel of antibodies against the 40-56, 89-104, 98-113 and 112-115 sequences of the prion protein, similarly to the abnormal prion protein extracted from scrapie-infected sheep. Interestingly, monoclonal antibody 3F4 was found to react with these three proteins in Western blot.