Prion Infectivity and PrPBSE in the Peripheral and Central Nervous System of Cattle 8 Months Post Oral BSE Challenge

Abstract

After oral exposure of cattle with classical bovine spongiform encephalopathy (C-BSE), the infectious agent ascends from the gut to the central nervous system (CNS) primarily via the autonomic nervous system. However, the timeline of this progression has thus far remained widely undetermined. Previous studies were focused on later time points after oral exposure of animals that were already 4 to 6 months old when challenged. In contrast, in this present study, we have orally inoculated 4 to 6 weeks old unweaned calves with high doses of BSE to identify any possible BSE infectivity and/or PrP^BSE in peripheral nervous tissues during the first eight months post-inoculation (mpi). For the detection of BSE infectivity, we used a bovine PrP transgenic mouse bioassay, while PrP^BSE depositions were analyzed by immunohistochemistry (IHC) and by protein misfolding cyclic amplification (PMCA). We were able to show that as early as 8 mpi the thoracic spinal cord as well as the parasympathetic nodal ganglion of these animals contained PrP^BSE and BSE infectivity. This shows that the centripetal prion spread starts early after challenge at least in this age group, which represents an essential piece of information for the risk assessments for food, feed, and pharmaceutical products produced from young calves.

Keywords: BSE; PrPBSE; cattle; infectivity; peripheral and central nervous system; prion protein; protein misfolding cyclic amplification (PMCA).

Figure 1

PrP^BSE accumulation in the obex of positive control cattle. IHC results obtained for the obex from positive controls IC 01 (36 mpi; (A)) and IC 04 (35 mpi; (B)); (A): a single neuron revealing a intracytoplasmatic and perineuronal staining pattern as well as moderate fine granular PrP^BSE accumulation in the neuropil of the dorsal motor nucleus of the vagus nerve (DMNV) of the obex from IC 01 (36 mpi); (B): intracytoplasmatic and perineuronal PrP^BSE accumulation as well as mild to severe fine to coarse granular accumulation of PrP^BSE in the neuropil of the DMNV of the obex from IC 04 (35 mpi); (A,B): Immunohistochemistry, PrP mAb 6C2, bar 50 µm.

Figure 2

PrP^BSE amplification by PMCA in the nodal ganglion at 8 months after challenge. From the third round of PMCA, seeding activity was shown in the nodal ganglion from animal IC 02 (8 mpi); this sample was analyzed in duplicate and subjected to 4 rounds of PMCA; M: marker.

Figure 3

PrP^BSE amplification by PMCA in the thoracic spinal cord from a calf at 8 months after challenge. PMCA detected PrP^BSE in the thoracic spinal cord segment T7 of IC 02 (8 mpi); (A): (+)++ positive PMCA reaction for the homogenate used for inoculation of bioassay mice; (B): analyzing a different location of the same T7 sample revealed amplification only in the third round (+ positive reaction); each sample was analyzed in duplicate and subjected to 3 rounds of PMCA. M: marker, R0: analyte homogenate diluted 1:10 in Tgbov XV brain substrate without sonication.

Figure 4

PrP^BSE accumulation in the coeliac and caudal mesenteric ganglion as well as the vagal nerve of positive control cattle. IHC results obtained for positive controls IC 01 (36 mpi; (A,D,G,J)) and IC 04 (35 mpi; (B,E,H,K)) as well as preclinical calf IC 03 (8 mpi; (C,F,I,L)); (A,B,D,E): fine to coarse granular intracytoplasmatic as well as perineuronal PrP^BSE accumulation in a ganglia cell of the coeliac ganglion (A,B) and the caudal mesenteric ganglion (D,E) of IC 01 (36 mpi; (A,D)) and IC 04 (35 mpi; (B,E)); H: intracytoplasmatic fine to coarse granular accumulation of PrP^BSE in a ganglia cell contained in the vagal nerve of IC 04 (35 mpi); (C,F,G,I–L): no staining reaction in samples from nervous tissues of calf IC 03 (8 mpi; (C,F,I,L)) as well as vagal nerve of IC 01 (G) and splanchnic nerves of IC 01 (J) and 04 (K), while the diffuse staining reaction in neural cells in the caudal mesenteric ganglion of IC 03 (F) represents an nonspecific background staining; (A–L): Immunohistochemistry, PrP mAb 6C2; (A,B,D,E,H): bar 20 µm; (C,F,G,I–L): bar 50 µm.

Figure 5

PrP^BSE amplification by PMCA in nervous tissue samples from positive control cattle. (A,B,D,E): Seeding activity was present in the coeliac ganglion of the positive controls IC 01 (36 mpi; (A)) and 04 (35 mpi; (B)) as shown by +++ (A) and ++ (B) positive PMCA reactions, while–in contrast to IHC–no PrP^BSE was detectable by PMCA in the caudal mesenteric ganglion of IC 01 (D) and 04 (E); (G,H,J,K): + positive PMCA reactions confirmed the presence of PrP^BSE in the parasympathetic vagal nerve (G,H) and sympathetic splanchnic nerve (J,K) of IC 01 (G,J) and 04 (H,K); (C,F,I,L): the mentioned peripheral ganglia and nerves of IC 03 (8 mpi) were negative by PMCA; All analyte tissue samples were analyzed in duplicate and subjected to 3 rounds (1st, 2nd, 3rd) of PMCA. An additional fourth round confirmed PrP^BSE amplification of weak positive results obtained with the initial PMCA run of some samples. M: marker, R0: analyte homogenate diluted 1:10 in Tgbov XV brain substrate without sonication.