Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Summary

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

Abstract

Infectious prion (PrP^Res) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases¹. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal^2,3 as well as from environmental sources^4-6. Scavengers and carnivores have potential to translocate PrP^Res material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrP^Res material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger⁷.

We describe procedures used to document passage of PrP^Res material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Introduction

Transmissible spongiform encephalopathies (TSE) are fatal infectious neurodegenerative disorders that affect wildlife, domestic animals, and humans. The infectious agent of TSE diseases appears to be misfolded or pathogenic isoforms (PrP^Res) of prion proteins¹. Animal TSE diseases include chronic wasting disease (CWD) in mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), elk (Cervus elaphus), and moose (Alces alces); scrapie in sheep and goats; bovine spongiform encephalopathy (BSE) in domestic cattle; transmissible mink encephalopathy in farmed mink; feline spongiform encephalopathy i....

Protocol

Our protocol is adapted from one we previously published⁷. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of the United States Department of Agriculture (USDA), Animal and Plant Health Inspection Service (APHIS), Wildlife Services (WS), National Wildlife Research Center (NWRC).

1. Crow Gavaging

1. Estimate passage time of 'pseudo brain material' through the alimentary tract of American crows.
   1. Mix 5 ml of cooked and scrambled whole egg with blue dye and gavage 1 crow using a 2 in gavage needle (Figure 1).
   2. Check crow eve....

Results

The procedures used demonstrate that the digestive system of crows does not eliminate PrP^Res infectivity 4 hr after oral gavage of scrapie brain homogenate⁷. All twenty crows that were gavaged with PrP^Res material subsequently transmitted PrP^Res material via feces to mice. Diseased mice were identified by manifestation of clinical mouse-scrapie signs and disease confirmation was completed by Western blot analysis.

Investigation of the retention time .......

Discussion

We demonstrate a procedure to document passage of PrP^Res material through the digestive system of crows. We used conventional methods to determine if crows have the ability to translocate PrP^Res material to disease-free geographic areas. Others have evaluated resistance of PrP^Res to ruminant^19-21 and rodent^22,23 digestive fluids, both of which failed to eliminate it. Future application of these techniques should be applied to other carnivores²⁴ as they cou.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RML Chandler strain mouse-adapted scrapie	Rocky Mountain Laboratories
RC57BL/6 mice	Hilltop Lab Animals
American crows	wild captured
Pen/Strep	Invitrogen	15140-122
Phosphate buffered Saline	Invitrogen	70011-044
Sonicator	Misonix
Proteinase-K solution	Roche	3115887001
Loading buffer	Invitrogen	NP0007 and 0009
Bis-tris SDS PAGE 12% gel	Invitrogen	NP0342
Immobilon PVDF membrane	Millipore	1SEQ00010
Tween 20	Sigma Aldrich	P2287
Bullet blender homogenizer	Braintree Scientific	BBX24B
2.3 mm Zirconia/silica beads	BioSpec Products	11079125Z
Bar224 anti-PrP monoclonal antibody	Cayman Chemical	10009035
Superblock	Thermo Scientific	37517
chemiluminescent substrate	Millipore	WBKLS0500
G-box gel documentation system	Syngene