Extraneural prion neuroinvasion without lymphoreticular system infection

Abstract

While prion infection of the lymphoreticular system (LRS) is necessary for neuroinvasion in many prion diseases, in bovine spongiform encephalopathy and atypical cases of sheep scrapie there is evidence to challenge that LRS infection is required for neuroinvasion. Here we investigated the role of prion infection of LRS tissues in neuroinvasion following extraneural inoculation with the HY and DY strains of the transmissible mink encephalopathy (TME) agent. DY TME agent infectivity was not detected in spleen or lymph nodes following intraperitoneal inoculation and clinical disease was not observed following inoculation into the peritoneum or lymph nodes, or after oral ingestion. In contrast, inoculation of the HY TME agent by each of these peripheral routes resulted in replication in the spleen and lymph nodes and induced clinical disease. To clarify the role of the LRS in neuroinvasion, the HY and DY TME agents were also inoculated into the tongue because it is densely innervated and lesions on the tongue, which are common in ruminants, increase the susceptibility of hamsters to experimental prion disease. Following intratongue inoculation, the DY TME agent caused prion disease and was detected in both the tongue and brainstem nuclei that innervate the tongue, but the prion protein PrP(Sc) was not detected in the spleen or lymph nodes. These findings indicate that the DY TME agent can spread from the tongue to the brain along cranial nerves and neuroinvasion does not require agent replication in the LRS. These studies provide support for prion neuroinvasion from highly innervated peripheral tissues in the absence of LRS infection in natural prion diseases of livestock.

FIG. 1.

Tissue distribution of PrP^Sc following inoculation of TME agents by neural and extraneural routes. Hamsters were inoculated with the HY TME or DY TME agent by either the intracerebral or intraperitoneal route. Hamsters were sacrificed after the onset of clinical disease and from clinically normal, aged DY TME agent-inoculated hamsters at greater than 500 days after intraperitoneal inoculation. The brain (Br), spleen (Sp), and submandibular lymph nodes (Sm) were collected for PrP^Sc analysis. Brain homogenates, containing 0.15 to 0.25 mg equivalents of tissue, and PrP^Sc-enriched preparations from the spleen and lymph nodes (25 mg equivalents) were prepared as described in the text and analyzed by SDS-PAGE and PrP Western blot. The positions of molecular mass markers are indicated to the left of the panel in kilodaltons.

FIG. 2.

Tissue distribution of PrP^Sc following intratongue inoculation of the HY TME and DY TME agents. Hamsters were inoculated in the tongue and sacrificed after the onset of clinical symptoms. The brain (Br), tongue (To), spleen (Sp), submandibular lymph node (Sm), cervical lymph node (Ce), and medial iliac lymph node (Mi) were collected and prepared for PrP^Sc analysis and Western blot as described for Fig. 1. Tissue amounts are indicated in milligram equivalents (Mg eq).

FIG. 3.

PrP^Sc deposition in the hypoglossal nucleus of hamsters following intratongue inoculation of the HY TME and DY TME agents. Hamsters were inoculated by the intratongue route with the HY TME agent (B) or DY TME agent (C). Brain was collected from HY TME- and DY TME agent-infected hamsters at 4 weeks (i.e., 35% of the HY TME agent incubation period had elapsed) and 23 weeks (i.e., 60% of the DY TME agent incubation period had elapsed) postinfection, respectively, and prepared for immunohistochemistry as described in Materials and Methods. PrP^Sc immunohistochemistry (red punctate signal) revealed staining in the hypoglossal nucleus that included intrasomata PrP^Sc deposits in motor neurons in HY TME agent infection and neuropil PrP^Sc deposits with a paucity of intrasomata staining in DY TME agent infection. Tissue was counterstained with hematoxylin (B and C). Nissl stain (A) of the hypoglossal nucleus from a mock-infected hamster illustrates somata of motor neurons. Arrowheads indicate hypoglossal motor neurons. Bar, 20 μm.