The authors have declared that no competing interests exist.
Conceived and designed the experiments: TAN TRS TG TR KV. Performed the experiments: TAN TRS TR KO JB MZ CMR CH BM RB. Analyzed the data: TAN JB KO GT MZ. Contributed reagents/materials/analysis tools: TAN TRS AB KO GT RB MZ KV EH. Wrote the paper: TAN TRS TR KO JB MZ KV.
Current address: Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, Washington, United States of America
Chronic wasting disease (CWD), the only known prion disease endemic in wildlife, is a persistent problem in both wild and captive North American cervid populations. This disease continues to spread and cases are found in new areas each year. Indirect transmission can occur via the environment and is thought to occur by the oral and/or intranasal route. Oral transmission has been experimentally demonstrated and although intranasal transmission has been postulated, it has not been tested in a natural host until recently. Prions have been shown to adsorb strongly to clay particles and upon oral inoculation the prion/clay combination exhibits increased infectivity in rodent models. Deer and elk undoubtedly and chronically inhale dust particles routinely while living in the landscape while foraging and rutting. We therefore hypothesized that dust represents a viable vehicle for intranasal CWD prion exposure. To test this hypothesis, CWD-positive brain homogenate was mixed with montmorillonite clay (Mte), lyophilized, pulverized and inoculated intranasally into white-tailed deer once a week for 6 weeks. Deer were euthanized at 95, 105, 120 and 175 days post final inoculation and tissues examined for CWD-associated prion proteins by immunohistochemistry. Our results demonstrate that CWD can be efficiently transmitted utilizing Mte particles as a prion carrier and intranasal exposure.
Chronic wasting disease (CWD) is a naturally occurring transmissible spongiform encephalopathy (TSE) of deer, elk and moose that affects captive as well as wild populations. Currently, 15 states, 2 Canadian provinces and South Korea have reported cases of CWD
Intranasal (IN) infection has been validated in rodent models by placing liquid inoculum into or in front of the nasal cavity
All procedures involving animals were performed to minimize suffering and were approved by the Institutional Animal Care and Use Committee at Colorado State University in accordance with the USDA Animal Welfare Act Regulation. CFR, title 9, chapter 1, subchapter A, parts 1–4.
Fifteen white-tailed deer (
At twelve weeks of age 12 deer were transported to a Colorado State University (CSU) BSL-2 facility for CWD-inoculation. The remaining two deer served as controls and remained at the USDA National Wildlife Research Center (NWRC) facility.
Blood was drawn from fawns upon arrival and sent to the USDA Agricultural Research laboratory in Pullman, WA for genetic analysis of the open reading frame of
A 20% homogenate of white-tailed deer brain from experimentally inoculated, CWD-terminal or CWD-negative deer was generated in 0.5× phosphate buffered saline (PBS) and glass beads utilizing a Blue Bullet homogenizer (Next Advance, Averill Park, NY) as previously described
We quantified the infectivity of our CWD+ inoculum with the cervid prion cell assay (CPCA) as described previously
Cells were passaged three times at four day intervals at 1∶4 and 1∶7 split ratios. When cells reached confluence at the third passage, 20,000 cells per well were filtered onto Multiscreen IP 96-well 0.45-µm filter plates (Elispot plates, Millipore, Billerica, MA). Plates were dried at 50°C and cells were digested for 90 min at 37°C in 60 µl of lysis buffer containing 5 µg/ml proteinase K (PK) then terminated with phenylmethanesulfonylfluoride (PMSF) (2 mM). To expose the epitope of PrP27-30, cells were incubated in 120 µl 3 M guanidinium thiocyanate in 10 mM Tris-HCl (pH 8.0) for 10 min at room temperature then rinsed four times with 160 µl PBS. For immunodetection, wells were filled with 120 µl of filtered 5% superblock (Pierce, Rockford, IL) and incubated for one hr at room temperature. The solution was removed by vacuum, and wells were incubated with 60 µl of 6H4 mAb, diluted 1: 5000 in TBST for one hr at RT or overnight at 4°C. Wells were rinsed four times with 160 µl of TBST then incubated with 60 µl AP-α-Mouse IgG (Southern Biotechnology Associates, Birmingham, AL), diluted 1: 5000 in TBST, after one hr at RT, the wells were rinsed four times with 160 µl TBST, followed by a final wash with PBS. Plates were allowed to dry completely. Visualization was done by adding 60 µl of AP conjugate substrate kit (Bio-Rad, Hercules, CA) at RT and rinsing twice with 160 µl water and allowed to completely dry. Images were scanned with a ImmunoSpot S6-V analyzer (Cellular Technology Ltd, Shaker Heights, OH), and spot numbers were determined using ImmunoSpot5 software (Cellular Technology Ltd, Shaker Heights, OH). Statistical analyses were performed using GraphPad Prism 5.0 for Mac OS X software. Prion titer was calculated in spot-forming CPCA units per gram of wet brain homogenate.
The deer were bottle-fed and hand-raised so they were easily manually restrained for the intranasal inoculations. The plastic transfer pipette containing the powdered inoculum was warmed to room temperature, inserted approximately 3 cm into the left nostril and inoculum puffed into the nasal cavity once a week for six weeks, exposing each deer to a total of 1.2 g (wet weight) of brain. Two mock-infected control deer were inoculated in the same fashion using CWD-negative brain/clay inoculum. Three deer were euthanized at each of three time points after the final inoculation: 95, 105, 120 days post inoculation (DPI); and two deer at 175 DPI. Because of the presence of genetic variation at codon 96, a GG, GS and SS deer was placed in each of the four time groups with the exception of the 105 DPI group, which contained one GG and two SS genotypes. The control deer were composed of one GG and one GS.
Two tracking methods were employed to determine the distribution of the CWD inoculum within nasal the cavity, both grossly and microscopically, after inoculation. To grossly determine the distribution of the clay inoculum, 500 µl of green fluorescent dye (GFD, Wizard Tattoo Ink, Chicago, IL) and 500 µl of deionized water were added to 250 mg of Mte clay and mixed thoroughly. The mixture was prepared and inoculated IN as described for brain homogenates. The ink was visible with both ultraviolet and visible light. One control deer was manually restrained and inoculated IN with GFD/Mte into the left nostril. Forty-five minutes later the deer was euthanized and necropsy conducted.
To track the uptake of the CWD/Mte material within the nasal mucosa, a lyophilized, fluorescently-labeled CWD prion was strategy employed. Purified deer CWD prion rods were isolated as previously described
Deer were sedated with Xylazine (Lloyd Laboratories, Shenandoah, IA) then euthanized via intravenous injection of Beuthanasia-D (Schering-Plough Animal Health Corp., Union, NJ). Deer were immediately transported to the Colorado State University Veterinary Diagnostic Laboratory for a complete post mortem examination.
At necropsy of the GFD/Mte tracer control deer, the nasal cavity, throat, and head lymph nodes were examined for the presence of GFD/Mte using a hand-held UV light source (Spectroline, Westbury, NY). Nasal turbinates were serially dissected and photographed. To detect microscopic-sized dyed particles, a fluorescent microscope was used with a UV excitation source and UV emission filter.
To visualize fluorescent prions, sections were counterstained with 1 µg/mL of the Carbocyanin lipophilic tracer DiOC18 and 100 ng/mL DAPI for 20 minutes, then mounted using ProLong Gold fluorescent mounting medium (Life Technologies, Madison, WI). Five to twelve 5 µm thick, paraffin-embedded tissue sections from nasal turbinates, retropharyngeal, submandibular and parotid lymph nodes and tonsils were viewed and photographed using an Olympus BX60 microscope (Center Valley, PA) equipped with a cooled charge-coupled diode camera.
Retropharyngeal, submandibular, parotid, pre-scapular, pre-femoral, mesenteric, and illiocecalcolic junction lymph nodes as well as tonsils, Peyer's patches, rectum, brain, and nasal tissues (cribriform plate, and ethmoid turbinates, two sections from each) were collected from the deer at necropsy and placed in 10% neutral buffered formalin for one week. The nasal tissues were then placed in 10% formic acid for three to five days for decalcification, trimmed, and embedded in paraffin blocks. Other tissues were trimmed and two to four sections of each were placed in plastic cassettes and allowed to fix for an additional two days. Slides were prepared as previously described for visualization and evaluation
The CWD status of the negative inoculum was verified by PMCA and the positive by western blot analysis (data not shown) prior to inoculation. Control brain inoculum remained negative after six 24 hr rounds of PMCA amplification and western blot visualization. Positive brain inoculum was positive on western blot visualization. The deer CWD prion infectivity titer was determined by CPCA using a highly sensitive Deer 5E9-S1 cell line (
We observed no clinical signs characteristic of CWD and no detectable PrPCWD by IHC in the obex, thalamus, hypothalamus, basal ganglia, olfactory bulb, or hippocampus of the brain in any of the deer at any of the time points. We next investigated subclinical infection by searching for PrPCWD in lymph nodes that drain or are proximal to the nasal cavity; retropharyngeal, submandibular, parotid, and palatine tonsil, as well as distal lymph nodes, pre-scapular, pre-femoral, mesenteric, Peyer's patches and the recto-anal mucosa associated lymphoid tissue (RAMALT). PrPCWD was not detected in either of the two mock-infected control deer (0/2 at 175 DPI), but was detected in lymph nodes of 10/11 of the CWD-inoculated deer by IHC (
No significant difference was detected in the number of affected follicles between the groups (ANOVA,
Codon 96 | DPI | Number of CWD+ follicles | % of affected |
genotype | total follicles | follicles | |
GG | 175 | 0/2547 | 0% |
GS | 175 | 0/2900 | 0% |
GS | 95 | 663/2141 | 31% |
GG | 95 | 1376/1972 | 70% |
SS | 95 | 66/2939 | 2% |
GG | 105 | 828/1635 | 51% |
SS | 105 | 0/2338 | 0% |
SS | 105 | 20/3069 | <1% |
GG | 120 | 1751/2396 | 73% |
GS | 120 | 155/1763 | 9% |
SS | 120 | 168/2061 | 8% |
GG | 175 | 467/2790 | 17% |
GS | 175 | 471/1790 | 26% |
Between 1600 and 3069 lymphoid follicles from head LNs, tonsils, pre-scapular and femoral LNs, Peyer's patches, mesenteric LN, gut LNs and rectum were evaluated in each deer for the presence of CWD by IHC. Percentage of total follicles IHC-positive for CWD ranged between 0->70%, depending on genotype. (
Upon genotypic analysis, it was discovered that although all deer were homozygous at codon 95 (QQ), all three genotypes (GG, GS, and SS) were represented at codon 96. We therefore sampled deer from each genotype at each time point. We found that the genotype at codon 96 affected PrPCWD temporal distribution and proportion of PrPCWD positive follicles within the lymphoid system (
The GG genotype had a wider PrPCWD distribution than GS and SS. *Tissue CWD % was significantly less than the GG genotype. ♦ Tissue CWD % was significantly less than the GS genotype. (Students one tailed T-test,
To determine if the deposition of the initial inocula could influence later replication sites and to understand the deposition and initial contact/entry sites for prions and the Mte within the nasal cavity, we sought to track the location of inocula immediately after exposure. We first puffed GFD/Mte dust (
6A. Longitudinal cut of a deer head without GFD/Mte under a blacklight. Inset- dissected nasal turbinates. 6B. Longitudinal cut of a deer head inoculated with GFD/Mte under a black light with visible GFD/Mte deposition. Insets- dissected nasal turbinates 6C. Ethmoid nasal turbinates under a blacklight. Arrows and circles indicate small particles of GFD/Mte. 6D. Mounted nasal turbinate with 100× magnification showing GFD/Mte particles associated with the nasal mucosa under normal light and with a ultraviolet filter. OE-Olfactory epithelium, NC-Nasal Cavity. Arrows indicate GFD/Mte (6E).
Highly enriched, fluorescently labeled prions were mixed with Mte, lyophilized, pulverized and puffed into the nasal cavity. (A) After 45 minutes, florescent prion aggregates (red) can be seen on (white arrowheads) and within the olfactory epithelium (OE) of the nasal turbinates. Tissue sections are counterstained with DiOC18 fluorescent membrane dye (green) and the nuclear stain DAPI (blue). A small proportion of prions can be seen near serous cells of the Bowman's glands (BG) in the lamina propria. (B) By 60 min, significant amount of prions were found in the lamina propria, with some aggregates (arrowhead) associated with nerve fibers (NF) emanating from the OE. (C) We detected no red signal from negative control sections from mock-inoculated deer. (D–G) Higher magnification of a Bowman's gland stained with DAPI (D), DiOC18 (E) and decorated with prions (F) that appear to localize on serous cells (G). NB, nerve bundle; scale bar, 20 µm.
We detected no prions associated with blood or lymphatic vessels in the lamina propria, or in any of the lymph nodes examined, including the retropharyngeal, submandibular and parotid lymph nodes, and palatine tonsils at these very early time points.
Indirect environmental CWD transmission has been demonstrated and several infectious tissues and excreta have been identified that could contaminate forage, soil and water
The olfactory bulb was of particular interest in this study. There are millions of olfactory sensory neurons (OSN) present in the nasal mucosa that connect directly to the olfactory bulb
In contrast to the brain, we found substantial PrPCWD in the lymph nodes and tonsils of the head (retropharyngeal, submandibular and parotid lymph nodes) in all genotypes. The tonsil findings are in agreement with those of a concurrent study using aerosolization of liquid inoculum
Dust can be an environmentally realistic, chronic, low dose mode of intranasal CWD exposure for deer and elk in the wild, particularly in dry western states such as Colorado and Wyoming. The size of dust particles is directly proportional to how far the particles are able to travel into the respiratory tract, with particles ≤1 µm able to travel into the deepest regions of the lung, 1–5 µm into the bronchiolar region, 5 µm into the upper lung and >10 µm caught in the nose and oral pharynx
We detected fluorescent prions on and within the olfactory epithelium and associated with nerve fibers and serous cells of the Bowman's glands in the lamina propria within one hr of exposure. We hypothesize that PrPCWD traffics from the nasal mucosa to draining lymph nodes via immune cells, or autonomously through lymphatic drainage, or both
Peyer's patches in the gut and the nasal mucosa contain dendritic cells (DC) and specialized membranous (M) cells
This study provided a unique opportunity to observe the effect of the dimorphism at codon 96 on IN infection of deer with Mte-adsorbed prion particulate. Several studies examining the prevalence of codon 96 polymorphisms have been done on wild populations with the GG polymorphism being the most common (72%), followed by GS (13–25%), and SS (3%)
The results of this study confirm that CWD can be successfully transmitted IN as a lyophilized prion particulate adsorbed to Mte and that genotype at codon 96 affects the lymphoid distribution of CWD within the body. Additionally, two novel intranasal tracking methods were employed that provided insight into CWD translocation within the nasal cavity. The data collected in this study may also shed light on why there is a higher prevalence of CWD in males, as males participate in more behaviors that generate dust. We propose chronic, long-term exposure to CWD prions adsorbed to dust particles to be a natural CWD infection route in addition to chronic oral and nasal contact exposure.
We thank Cody Minor, Nikki Crider, Jessie Gorges and Kiera Wood for their excellent care of the fawns, Dr. Pauline Nol for fawn veterinary care, Karl Held for his assistance in maintaining the animal facilities, other NWRC staff that assisted in pen construction, Dana Hill for sample handling and Angela Bosco-Lauth, Airn Tolnay and Paul Gordy for animal care at CSU and William O'Connor for proof-reading. Additionally, we would like to thank the USDA Veterinary Services' Dr. Jack Rhyan and Matt McCollum for the use of their animal facilities and the Colorado Division of Wildlife for transport/import permits.