PrPC expression and prion seeding activity in the alimentary tract and lymphoid tissue of deer
RESEARCH ARTICLE
PrPC expression and prion seeding activity in the alimentary tract and lymphoid tissue of deer
Kristen A. Davenport, Clare E. Hoover, Jifeng Bian, Glenn C. Telling, Candace K. Mathiason, Edward A. Hoover Published: September 7, 2017https://doi.org/10.1371/journal.pone.0183927
Abstract
The agent responsible for prion diseases is a misfolded form of a normal protein (PrPC). The prion hypothesis stipulates that PrPC must be present for the disease to manifest. Cervid populations across the world are infected with chronic wasting disease, a horizontally-transmissible prion disease that is likely spread via oral exposure to infectious prions (PrPCWD). Though PrPCWD has been identified in many tissues, there has been little effort to characterize the overall PrPC expression in cervids and its relationship to PrPCWD accumulation. We used immunohistochemistry (IHC), western blot and enzyme-linked immunosorbent assay to describe PrPC expression in naïve white-tailed deer. We used real-time, quaking-induced conversion (RT-QuIC) to detect prion seeding activity in CWD-infected deer. We assessed tissues comprising the alimentary tract, alimentary-associated lymphoid tissue and systemic lymphoid tissue from 5 naïve deer. PrPC was expressed in all tissues, though expression was often very low compared to the level in the CNS. IHC identified specific cell types wherein PrPC expression is very high. To compare the distribution of PrPC to PrPCWD, we examined 5 deer with advanced CWD infection. Using RT-QuIC, we detected prion seeding activity in all 21 tissues. In 3 subclinical deer sacrificed 4 months post-inoculation, we detected PrPCWD consistently in alimentary-associated lymphoid tissue, irregularly in alimentary tract tissues, and not at all in the brain. Contrary to our hypothesis that PrPC levels dictate prion accumulation, PrPC expression was higher in the lower gastrointestinal tissues than in the alimentary-associated lymphoid system and was higher in salivary glands than in the oropharyngeal lymphoid tissue. These data suggest that PrPC expression is not the sole driver of prion accumulation and that alimentary tract tissues accumulate prions before centrifugal spread from the brain occurs.
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Our data demonstrate that PrPC expression is widespread in white-tailed deer and confirms that symptomatic, CWD-positive, white-tailed deer have widespread prion seeding activity. Several questions arise from this work: 1) What is the role of local, cellular PrPC in the generation of prions that are excreted by infected cervids? CWD prions have been detected in a number of excreta from cervids [30, 41, 42, 63–66] and in the organs that generate those excreta [30], but we are left to wonder whether the misfolding event that produced those prions occurred in the excretory organ, or whether the prions were transported there. 2) Can many organs be used for CWD diagnosis in deer? Our data suggest that RT-QuIC may permit the testing of a broad array of tissues and that many tissues have detectable prion seeding activity later in disease progression. 3) What mechanism explains the presence of prion seeding activity in alimentary tissues before neuroinvasion? Our data suggest that prions replicate in tissues of the alimentary tract before they reach the brain. This work contributes to the growing body of evidence that CWD prions are widespread within cervids and that PrPC expression alone does not dictate the kinetics of prion spread in the body.
P6 Pathways of prion spread during early chronic wasting disease in deer
Dr Clare Hoover1, Ms Kristen Davenport1, Dr Davin Henderson1, Dr Nathaniel Denkers1, Dr Candace Mathiason1, Dr Claudio Soto2, Dr Mark Zabel1, Dr Edward Hoover1
1Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, United States, 2Department of Neurology, University of Texas, Houston, United States
Aims: Among prion infections, two scenarios of prion spread are generally observed: (a) early lymphoid tissue replication or (b) direct neuroinvasion without substantial prior lymphoid amplification. In nature, cervids are infected with chronic wasting disease by oral and nasal mucosal exposure, and studies of early CWD pathogenesis have implicated pharyngeal lymphoid tissue as the earliest sites of prion accumulation. However, knowledge of chronological events of prion spread during early CWD infection remains incomplete.
Methods: To investigate early CWD pathogenesis, we exposed white-tailed deer to CWD prions by mucosal routes and performed serial necropsies during the initial four months post-exposure to assess PrP-CWD tissue distribution. Because prion burdens during the initial stages of infection can be low, real-time quaking-induced conversion (RT-QuIC), end-point quaking-induced conversion (EP-QuIC) and tyramide-signal amplification immunohistochemistry (TSA-IHC) methods were used to enhance PrP-CWD detection.
Results: Although PrP-CWD was not detected by either method in the initial days (1 and 3) post-exposure, we observed PrP-CWD seeding activity and follicular immunoreactivity in oropharyngeal lymphoid tissues (tonsil, retropharyngeal, mandibular, and parotid lymph nodes) at 1 and 2 months post-exposure (MPE). At 3 MPE, PrP-CWD replication had expanded to all systemic lymphoid tissues. By 4 MPE, the PrP-CWD burden in all lymphoid tissues had increased, and approached levels observed in terminal disease, yet there was no evidence of nervous system invasion. EP-QuIC evaluation of all necropsy tissues collected at 4 MPE identified prion seeding activity in parotid and mandibular salivary glands, in addition to all systemic lymphoid tissues.
Conclusions: These results identify the oropharynx as the first site of CWD prion entry and that the initial phase of prion amplification occurs in the oropharyngeal lymphoid tissues followed by rapid dissemination to systemic lymphoid tissues. This lymphoid replication phase appears to precede neuroinvasion or the establishment of a chronic sub-clinical carrier state. The identification of CWD prion seeding activity in salivary glands suggests that saliva is the earliest source of excreted prions and environmental contamination.
Experimental transmission of the chronic wasting disease agent to swine after oral or intracranial inoculation
S. Jo Moore1,2, M. Heather West Greenlee3, Naveen Kondru3, Sireesha Manne3, Jodi D. Smith1, Robert A. Kunkle1, Anumantha Kanthasamy3 and Justin J. Greenlee1* + Author Affiliations
1Virus and Prion Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, Ames, Iowa, United States of America 2Oak Ridge Institute for Science and Education, Oak Ridge, Tennessee, United States of America 3Department of Biomedical Sciences, Iowa State University College of Veterinary Medicine, Ames, Iowa, United States of America
ABSTRACT
Chronic wasting disease (CWD) is a naturally occurring, fatal neurodegenerative disease of cervids. The potential for swine to serve as a host for the agent of chronic wasting disease is unknown. The purpose of this study was to investigate the susceptibility of swine to the CWD agent following experimental oral or intracranial inoculation. Crossbred piglets were assigned to one of three groups: intracranially inoculated (n=20), orally inoculated (n=19), or non-inoculated (n=9). At approximately the age at which commercial pigs reach market weight, half of the pigs in each group were culled (‘market weight’ groups). The remaining pigs (‘aged’ groups) were allowed to incubate for up to 73 months post inoculation (MPI). Tissues collected at necropsy were examined for disease-associated prion protein (PrPSc) by western blotting (WB), antigen-capture immunoassay (EIA), immunohistochemistry (IHC) and in vitro real-time quaking induced conversion (RT-QuIC). Brain samples from selected pigs were also bioassayed in mice expressing porcine prion protein. Four intracranially inoculated aged pigs and one orally inoculated aged pig were positive by EIA, IHC and/or WB. Using RT-QuIC, PrPSc was detected in lymphoid and/or brain tissue from one or more pigs in each inoculated group. Bioassay was positive in 4 out of 5 pigs assayed. This study demonstrates that pigs can support low-level amplification of CWD prions, although the species barrier to CWD infection is relatively high. However, detection of infectivity in orally inoculated pigs using mouse bioassay raises the possibility that naturally exposed pigs could act as a reservoir of CWD infectivity.
IMPORTANCE We challenged domestic swine with the chronic wasting disease agent by inoculation directly into the brain (intracranially) or by oral gavage (orally). Disease-associated prion protein (PrPSc) was detected in brain and lymphoid tissues from intracranially and orally inoculated pigs as early as 8 months of age (6 months post-inoculation). Only one pig developed clinical neurologic signs suggestive of prion disease. The amount of PrPSc in the brains and lymphoid tissues of positive pigs was small, especially in orally inoculated pigs. Regardless, positive results in orally inoculated pigs suggest that it may be possible for swine to serve as a reservoir for prion disease under natural conditions.
FOOTNOTES
↵*Corresponding author: Email: justin.greenlee@ars.usda.gov This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
Research Project: TRANSMISSION, DIFFERENTIATION, AND PATHOBIOLOGY OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES
Location: Virus and Prion Research
Title: Disease-associated prion protein detected in lymphoid tissues from pigs challenged with the agent of chronic wasting disease
Author item Moore, Sarah item Kunkle, Robert item Kondru, Naveen item Manne, Sireesha item Smith, Jodi item Kanthasamy, Anumantha item West Greenlee, M item Greenlee, Justin
Submitted to: Prion Publication Type: Abstract Only Publication Acceptance Date: 3/15/2017 Publication Date: N/A Citation: N/A Interpretive Summary:
Technical Abstract: Aims: Chronic wasting disease (CWD) is a naturally-occurring, fatal neurodegenerative disease of cervids. We previously demonstrated that disease-associated prion protein (PrPSc) can be detected in the brain and retina from pigs challenged intracranially or orally with the CWD agent. In that study, neurological signs consistent with prion disease were observed only in one pig: an intracranially challenged pig that was euthanized at 64 months post-challenge. The purpose of this study was to use an antigen-capture immunoassay (EIA) and real-time quaking-induced conversion (QuIC) to determine whether PrPSc is present in lymphoid tissues from pigs challenged with the CWD agent.
Methods: At two months of age, crossbred pigs were challenged by the intracranial route (n=20), oral route (n=19), or were left unchallenged (n=9). At approximately 6 months of age, the time at which commercial pigs reach market weight, half of the pigs in each group were culled (<6 challenge="" groups="" month="" pigs="" remaining="" the="">6 month challenge groups) were allowed to incubate for up to 73 months post challenge (mpc). The retropharyngeal lymph node (RPLN) was screened for the presence of PrPSc by EIA and immunohistochemistry (IHC). The RPLN, palatine tonsil, and mesenteric lymph node (MLN) from 6-7 pigs per challenge group were also tested using EIA and QuIC.
Results: PrPSc was not detected by EIA and IHC in any RPLNs. All tonsils and MLNs were negative by IHC, though the MLN from one pig in the oral <6 5="" 6="" at="" by="" detected="" eia.="" examined="" group="" in="" intracranial="" least="" lymphoid="" month="" months="" of="" one="" pigs="" positive="" prpsc="" quic="" the="" tissues="" was="">6 months group, 5/6 pigs in the oral <6 4="" and="" group="" months="" oral="">6 months group. Overall, the MLN was positive in 14/19 (74%) of samples examined, the RPLN in 8/18 (44%), and the tonsil in 10/25 (40%). Conclusions:
This study demonstrates that PrPSc accumulates in lymphoid tissues from pigs challenged intracranially or orally with the CWD agent, and can be detected as early as 4 months after challenge.
CWD-infected pigs rarely develop clinical disease and if they do, they do so after a long incubation period. This raises the possibility that CWD-infected pigs could shed prions into their environment long before they develop clinical disease.
Furthermore, lymphoid tissues from CWD-infected pigs could present a potential source of CWD infectivity in the animal and human food chains.
CONFIDENTIAL
EXPERIMENTAL PORCINE SPONGIFORM ENCEPHALOPATHY
While this clearly is a cause for concern we should not jump to the conclusion that this means that pigs will necessarily be infected by bone and meat meal fed by the oral route as is the case with cattle. ...
we cannot rule out the possibility that unrecognised subclinical spongiform encephalopathy could be present in British pigs though there is no evidence for this: only with parenteral/implantable pharmaceuticals/devices is the theoretical risk to humans of sufficient concern to consider any action.
Our records show that while some use is made of porcine materials in medicinal products, the only products which would appear to be in a hypothetically ''higher risk'' area are the adrenocorticotrophic hormone for which the source material comes from outside the United Kingdom, namely America China Sweden France and Germany. The products are manufactured by Ferring and Armour. A further product, ''Zenoderm Corium implant'' manufactured by Ethicon, makes use of porcine skin - which is not considered to be a ''high risk'' tissue, but one of its uses is described in the data sheet as ''in dural replacement''. This product is sourced from the United Kingdom.....
snip...see much more here ;
WEDNESDAY, APRIL 05, 2017
Disease-associated prion protein detected in lymphoid tissues from pigs challenged with the agent of chronic wasting disease
TUESDAY, APRIL 18, 2017
*** EXTREME USA FDA PART 589 TSE PRION FEED LOOP HOLE STILL EXIST, AND PRICE OF POKER GOES UP ***
TUESDAY, SEPTEMBER 12, 2017
CDC Now Recommends Strongly consider having the deer or elk tested for CWD before you eat the meat
kind regards, terry
posted by Terry S. Singeltary Sr. at
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