The Animal Ethics Committee and Animal Care and Use Committee of the National Institute of Animal Health approved the study. Five Holstein calves 2–3 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). A wide range of tissues was sampled at subsequent necropsy. 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 μg 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.

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). Control mice showed no apparent abnormality >500 days 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.