The test results showed that nearly all of the cattle (40/46) from 1 mpi to 44 mpi carried BSE infectivity and/or detectable amounts of PrPSc in their small intestines. We were able to demonstrate infectivity not only in the ileum, but also in the ileocaecal junction and in the jejunum and in some animals in all three anatomical locations simultaneously.
Samples for biochemical examinations and the bioassay were taken from one PP allowing a direct comparison of these methods. Clear differences in sensitivities were observed. While the BioRad TeSeE failed to detect any amount of PrPSc, both the IDEXX HerdChek and the PTA-immunoblot were positive in a number of samples from the ileum/ileocaecal-junction. The most sensitive test, however, was the bioassay, using bovine PrP over-expressing transgenic mice (Tgbov XV), showing infectivity in all parts of the small intestine. The highest amounts of infectivity (high transmission rates, short incubation periods) were found in the ileum, a moderate degree in the ileocaecal junction and traces of infectivity in the jejunum. A high sensitivity was also achieved through IHC, using a second PP. Nearly all animals examined (40/46) revealed detectable amounts of PrPSc mainly in the ileum but also in the ileocaecal-junction. In an earlier study only approximately one third of the cattle examined showed positive results in the distal ileum . These differences in sensitivity are most probably due to the larger number of sections prepared in our study. For example the total number of follicles examined here is 3-5 times higher than in comparable studies and the same is true for the plexus numbers. Due to the higher number of samples examined and therefore a higher sensitivity, this allowed us not only the very early detection of PrPSc in the ENS of a cow (IT28) at 16 mpi, but also we were able to demonstrate PrPSc in the ENS of one cow (IT26) which we reported to be negative two years before .
The results presented here add new insights into the gut-associated pathogenesis of BSE in cattle which shows a clear time-dependent pattern. It has been shown for some sheep scrapie infections that a latent period of at least about one month after exposure occurs during which no detectable infection is present [9, 22, 23]. This time lag is supposedly due to insufficient amounts of PrPSc for detection by immunohistochemistry [9, 22]. A similar time-dependent pattern for the detection of PrPSc is seen in the cattle examined here. However, the time lag between replication of the agent and accumulation of PrPSc in detectable amounts by IHC seems to be approximately four months. A shorter time-lag seems unlikely due to the only very weak amounts of PrPSc in ileal follicles at 4 mpi. During the first 8 mpi of BSE infection in cattle there seems to be an equilibrium between replication, accumulation and degradation as indicated by weak amounts of PrPSc represented by a punctate reaction pattern in TBM's. As the incubation time increased, the balance changed in favour of a massive accumulation of PrPSc in follicles of the PP's as seen at 12 mpi. Corresponding results are seen with the bioassay since younger animals up to 12 mpi showed a wide distribution of infectivity affecting all localizations which indicates a multifocal increase of the BSE agent. Infectivity data for the distal ileum presented here are compatible with those reported in UK studies [3, 4, 24]. For the distal ileum a peak at 12 mpi concerned both the number of follicles involved and the amount of PrPSc detectable in the individual follicles. Most interestingly, PrPSc is detectable in FDC as well as in TBM, indicating increased clearance activities by the latter, as described already for scrapie [13, 25]. The very low (if any) PrPSc accumulation and infectivity loads seen at time points 16 mpi and 20 mpi also support this clearance hypothesis. Such a decrease in the infectivity loads in ileal PP's of BSE-infected cattle in 20 mpi animals has also been described before .
A second peak of PrPSc accumulation, involving both TBM's and FDC was seen at 24 mpi, suggesting a replication cycle (i.e. a shift of the balance between accumulation and degradation in favour of accumulation) for BSE about 12 mpi. Infectivity studies done previously using BoPrP-Tg110 mice showed/revealed a comparable low incubation period of 24 mpi for 1 mouse out of 5 mice . Therefore infectivity data from that time point would be of interest, and will be done in future studies. A third peak of PrPSc accumulation in the ileal PP's can be demonstrated, after a clear decrease at 28 mpi, in single cows at 32, 36 and 40 mpi, reflecting an individual variability in older cows.
The exact route of infection for the ENS, which is thought to be the entry point to the peripheral nervous system, still remains unclear . In particular during the pathogenesis of BSE an accumulation of PrPSc in the ENS has been rarely reported and was confined to clinical cases [3, 6]. In contrast, in sheep scrapie a wide distribution of PrPSc in the ENS occurs, often associated with abundant deposits of PrPSc in the adjacent PP's [14, 28] indicating an infection of the ENS via the GALT. However, a direct route via nerve fibres underneath the villous epithelium is also discussed [9, 15]. In the study presented here evidence for both routes of infection can be seen. We observed one cow at 12 mpi which had a slight staining reaction in the submucosa directly adjacent to a follicle, which might reflect an early neuro-invasion of a submucosal plexus. Unfortunately we were not able to clearly characterize the cells involved and macrophages penetrating the submucosa can not completely be ruled out. The first clear evidence for an ENS infection is seen in a single myenteric plexus at 16 mpi without adjacent follicles staining. Moreover, it is striking that in younger cows the accumulations of PrPSc were confined to single plexuses, whereas later in the incubation period, in particular associated with end-stage BSE, the number of affected plexuses increased. However, in most of the samples examined a randomly distributed pattern of affected myenteric plexuses is obvious, a clear association to affected follicles is not seen suggesting a direct neuro-invasion without an involvement of the GALT. Nevertheless these results clearly indicate that the ENS is involved in the early propagation of PrPSc, but to a much lesser extend as compared to scrapie. Furthermore, the marginally higher number of affected plexus seen in clinical cases in combination with their multifocal distribution pattern could be due to a local accumulation over time, but a limited spread along the ENS cannot be completely ruled out.
Our presented data are relevant for a risk assessment based SRM definition. At least in younger animals up to 12 mpi the jejunum carries BSE infectivity. A recently published quantitative study reported a considerable amount of lymphoid and neural tissues in natural sausage casing produced from cattle small intestine after the cleaning procedure . Cross-contaminations between animals of various ages during the production process as well as during food preparation cannot be ruled out. Moreover, it has to be emphasized that only a very small proportion of the jejunum, (which has an overall length of 25-30 m and contains up to 40 PP) was examined in this current study.
In summary our study shows that nearly all of the BSE-infected cattle that were examined had BSE infectivity and/or detectable amounts of PrPSc in their small intestines. The highest amounts of BSE infectivity and/or PrPSc were seen in the ileum and ileocaecal junction with lower amounts in the jejunum. Clear time-dependent variations in the detectable amounts of PrPSc were visible. Younger cattle killed 8 mpi to 12 mpi carried higher PrPSc levels and had a more widespread distribution of PrPSc than animals at 16 mpi to 20 mpi. The data from this study show a more widespread distribution of the BSE agent in the small intestines in particular of pre-clinical cattle and are the first describing infectivity in jejunal samples of cattle. Therefore the data presented here are important for the definition of SRM and the implementation of their removal as part of functional public health protection measures against BSE.