Further preliminary observations are reported of an experiment to examine the spread of infectivity and the occurrence of pathological changes in cattle exposed orally to infection with bovine spongiform encephalopathy. Calves were dosed at four months of age and clinically monitored groups were killed sequentially from two to 40 months after inoculation. Tissues were collected for bioassay, for histopathological examinations and for the detection of PrP. Previous reported observations have included the presence of infectivity in the distal ileum of cattle killed after six to 18 months, the earliest onset of clinical signs in an exposed animal after 35 months, and diagnostic histopathological changes in the brain, in association with clinical disease, after 36, 38 and 40 months. In spite of the relative inefficiency of the bioassay of scrapie-like agents across a species barrier the new observations confirm that the onset of clinical signs and pathological changes in the central nervous system (CNS) occur at approximately the same time. The earliest pathological change, the presence of abnormal PrP 32 months after inoculation, coincided with the earliest detected infectivity in the CNS and occurred shortly before there was evidence of typical spongiform changes in the brain 36 months after inoculation. Infectivity has now been demonstrated in the peripheral nervous system, in the cervical and thoracic dorsal root ganglia 32 to 40 months after inoculation and in the trigeminal ganglion 36 and 38 months after inoculation. At the time of writing evidence of infectivity in other tissues is confined to the distal ileum, not only after six to 18 months but also after 38 and 40 months, but these findings may be supplemented by the results of further mouse assays. Nevertheless, they are in general agreement with current knowledge of the pathogenesis of scrapie.

To determine the mechanisms of intestinal transport of infection, and early pathogenesis, of sheep scrapie, isolated gut-loops were inoculated to ensure that significant concentrations of scrapie agent would come into direct contact with the relevant ileal structures (epithelial, lymphoreticular, and nervous). Gut loops were inoculated with a scrapie brain pool homogenate or normal brain or sucrose solution. After surgery, animals were necropsied at time points ranging from 15 min to 1 month and at clinical end point. Inoculum-associated prion protein (PrP) was detected by immunohistochemistry in villous lacteals and in sub-mucosal lymphatics from 15 min to 3.5 h post-challenge. It was also detected in association with dendritic-like cells in the draining lymph nodes at up to 24 h post-challenge. Replication of infection, as demonstrated by the accumulation of disease-associated forms of PrP in Peyer's patches, was detected at 30 days and sheep developed clinical signs of scrapie at 18-22 months post-challenge. These results indicate discrepancies between the routes of transportation of PrP from the inoculum and sites of de novo-generated disease-associated PrP subsequent to scrapie agent replication. When samples of homogenized inoculum were incubated with alimentary tract fluids in vitro, only trace amounts of protease-resistant PrP could be detected by western blotting, suggesting that the majority of both normal and abnormal PrP within the inoculum is readily digested by alimentary fluids.

BackgroundThe majority of atypical bovine spongiform encephalopathy (BSE) cases so far identified worldwide have been detected by active surveillance. Consequently the volume and quality of material available for detailed characterisation is very limiting. Here we report on a small transmission study of both atypical forms, H- and L-type BSE, in cattle to provide tissue for test evaluation and research, and to generate clinical, molecular and pathological data in a standardised way to enable more robust comparison of the two variants with particular reference to those aspects most relevant to case ascertainment and confirmatory diagnosis within existing regulated surveillance programmes.ResultsTwo groups of four cattle, intracerebrally inoculated with L-type or H-type BSE, all presented with a nervous disease form with some similarities to classical BSE, which progressed to a more dull form in one animal from each group. Difficulty rising was a consistent feature of both disease forms and not seen in two BSE-free, non-inoculated cattle that served as controls. The pathology and molecular characteristics were distinct from classical BSE, and broadly consistent with published data, but with some variation in the pathological characteristics. Both atypical BSE types were readily detectable as BSE by current confirmatory methods using the medulla brain region at the obex, but making a clear diagnostic distinction between the forms was not consistently straightforward in this brain region. Cerebellum proved a more reliable sample for discrimination when using immunohistochemistry.ConclusionsThe prominent feature of difficulty rising in atypical BSE cases may explain the detection of naturally occurring cases in emergency slaughter cattle and fallen stock. Current confirmatory diagnostic methods are effective for the detection of such atypical cases, but consistently and correctly identifying the variant forms may require modifications to the sampling regimes and methods that are currently in use.

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This study examines tissues from sequential-kill, time-course pathogenesis studies to refine estimates of the age at which disease-specific PrP (PrP Sc ) can first be detected in the central nervous system (CNS) and related peripheral nervous system ganglia of cattle incubating bovine spongiform encephalopathy (BSE). Such estimates are important for risk assessments of the age at which these tissues should be removed from cattle at slaughter to prevent human and animal exposure to BSE infection. Tissues were examined from cattle dosed orally with 100 or 1 g BSE-infected brain. Incubation period data for the doses were obtained from attack rate and the sequential-kill studies. A statistical model, fitted by maximum likelihood, accounted for the differences in the lognormal incubation period and the logistic probability of infection between different dose groups. Initial detection of PrP Sc during incubation was invariably in the brainstem and the earliest was at 30 and 44 months post-exposure for the 100 g-and 1 g-dosed sequential-kill study groups, respectively. The point at which PrP Sc in 50 % of the animals would be detected by immunohistochemistry applied to medulla-obex was estimated at 9.6 and 1.7 months before clinical onset for the 100 g-and 1 g-dosed cattle, respectively, with a low probability of detection in any of the tissues examined at more than 12 months before clinical onset. PrP Sc was detected inconsistently in dorsal root ganglia, concurrent with or after detection in CNS, and not at all in certain sympathetic nervous system ganglia.