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From: TSS ()
Subject: Re: Infectious Prions in the Saliva and Blood of Deer with Chronic Wasting Disease
Date: November 1, 2006 at 9:16 am PST

In Reply to: Infectious Prions in the Saliva and Blood of Deer with Chronic Wasting Disease posted by TSS on October 5, 2006 at 1:45 pm:

www.sciencemag.org/

Supporting Online Material for

Infectious Prions in the Saliva and Blood of Deer with

Chronic Wasting Disease

Candace K. Mathiason, Jenny G. Powers, Sallie J. Dahmes, David A. Osborn,

Karl V. Miller, Robert J. Warren, Gary L. Mason, Sheila A. Hays, Jeanette Hayes-Klug,

Davis M. Seelig, Margaret A. Wild, Lisa L. Wolfe, Terry R. Spraker, Michael W. Miller,

Christina J. Sigurdson, Glenn C. Telling, Edward A. Hoover*

*To whom correspondence should be addressed. E-mail: edward.hoover@colostate.edu

Published 6 October, Science 314, 133 (2006)

DOI: 10.1126/science.1132661

This PDF file includes:

Materials and Methods

SOM Text

Tables S1 and S2

References

Supporting Online Material

SOM Materials and Methods

Immunoblotting methods: Tissue homogenates were prepared from the obex region of the

medulla oblongata encompassing the dorsal motor vagal nucleus. 20% w/v homogenates

were prepared in NP-40 buffer (10mM Tris-HCl buffer pH 7.5, 0.5% NP-40, 0.5%

sodium deoxycholate) by Fastprep TM disruption at setting 6.5 for 45 seconds. Twentyfive

µl of each homogenate was mixed with 5µl proteinase K to a final concentration of

20 µg/ml and incubated for 30 minutes at 37C with shaking. Proteinase K activity was

stopped with 4µl 200mM Pefablock and an equivalent protein concentration of each

sample was mixed with 10 µl sample buffer (Invitrogen-20% NP0009, 50% NP0007),

5µl NP-40 buffer, was heated to 95C for 5 minutes and underwent separation by 12%

Bis-Tris precast polyacrylamide gel electrophoresis (PAGE) (Invitrogen) at 150 volts for

2.5 hours in 1x MOPS (Invitrogen). PAGE separated proteins were transferred to

polyvinylidene fluoride (PVDF) membrane for 1 hour at 100 volts in transfer buffer

(0.025M Trizma base, 0.2M glycine, 20% methanol, pH 8.3). After the PVDF

membranes blocked overnight at room temperature in Pierce BlockerTM they were probed

with the PrP specific antibody BAR224 followed by hrp-goat anti-mouse IgG diluted in

BlockerTM. Membranes were washed for 1 hour after blocking and between antibodies

with wash buffer (0.1M Tris, 0.15M NaCl pH 8.0). To visualize PrP bands the PVDF

membranes were developed with the AmershamTM ECL detection system.

Immunohistochemistry methods: Ihc was performed by the Colorado State University

Veterinary Diagnostic Laboratory (CSUVDL) employing the VentanaTM Nexus

autostainer and Ventana TM PrPCWD specific antibody as described by Spraker et. al. (1).

SOM Text

Protective husbandry protocols: Exacting protocols were enacted to preclude extraneous

exposure or cross-contamination among cohorts and to protect personnel. These

protocols included shower-in procedures, the wearing of Tyvek™ clothing, face masks,

head covers, footwear, strict facility traffic flow, sourcing of feeds and bedding from

outside CWD infected regions, and animal-specific biopsy and sample collection

instruments. Sham-inoculated control deer (cohort #5) were housed in the same building

as sentinels to assure freedom from cross or adventitious contamination.

Cervid to cervid salivary interactions: Grooming interactions, shared water sources, salt

licks, scrape sites, and forage sites, especially those in which cervids are in greater

density, e.g. during the breeding season, low predation territories and captivity (e.g.

cervid farms), all would be expected to facilitate salivary cross contact.

Clinical signs: To detect and monitor clinical manifestations of CWD (2- 4) deer were

observed daily by project-dedicated caretakers intimately familiar with each animal.

Onset of subtle clinical signs consistent with CWD were detected at 15-20 months pi in 2

of 4 positive control animals (cohort # 4). The disease onset was manifest primarily by

perceived body muscle mass reduction and was measured by gradual weight loss, which

reached ≥20% of maximum body weight over 5 to 8 months. Additional clinical signs

included a rough-appearing hair coat due to piloerection and a body stance characterized

by a lower position of the head and a wider lateral separation of the limbs. Changes in

behavior included hyperphagia and polydipsia in the face of weight loss, a head tossing

motion, repetitive exaggerated lifting of the legs, diminished alertness, and occasionally

aggressive behavior in the advanced stage of disease. Animals were euthanized when

they displayed advanced clinical signs of CWD. Clinical signs were not observed in the

CWD+ deer from cohorts #1-#3, probably because these animals were electively

euthanized and necropsied at 18 months pi, although 7 of the 8 had PrPCWD demonstrable

in neural and lymphoid tissues at necropsy.

Table S1

Table S1. CWD bioassay inocula sources

Deer source Donor animal ID numbers

Brain Urine + Feces Saliva Blood

CSUVDL TS-989

NPS 0428 0428 0428

CDOW LA01 LA01/JA01 LA01/JA01 LA01

JA01/D10 JA01 33a02

UGA 1/2 1/2 1/2 1/2

CWD positive brain, blood, saliva, urine and feces from terminal free ranging mule deer were

provided by the Colorado State University Veterinary Diagnostic Laboratory (CSUVDL) and the

National Park Service (NPS). The Colorado Division of Wildlife (CDOW) provided similar

inocula from terminal captive mule deer. CWD negative control inocula source was provided by

the University of Georgia, Athens (UGA).

Table S2

Table S2. Deer cohort #1-#5 PRNP codon 96 genotype

Animal cohort Route of

inoculation

Donor ID No. PRNP 96 Months pi 1st

CWD+ status

Cohort #1-blood IP 33a02 G/G 18

blood IP 0428 G/G 12

blood IV LAO1 G/G 12

Cohort #2-saliva PO JA01 G/G 12

saliva PO JAO1/LAO1 G/G 18

saliva PO 0428 G/G 12

Cohort #3-urine+feces PO JAO1/D10 G/S Negative

urine+feces PO 0428 G/S Negative

urine+feces PO LAO1/JAO1 nda nd

Cohort #4-brain IC LAO1 G/S 12

brain IC TS-989 G/S 6b

brain PO LAO1 G/S 12b

brain PO TS-989 G/G 3

Cohort #5-negative All routes UGA 1/2 G/G Negative

negative All routes UGA 1/2 G/G Negative

All deer in cohorts #1-blood, #2-saliva and #5-negative control were PRNP 96 G/G. Three deer

in cohort #4-brain (n=2 IC and n=1 PO) were PRNP 96 G/S while n=1 PO was PRNP 96 G/G.

Both cohort #3 urine+feces deer were PRNP 96 G/S. aOne of 3 deer in cohort #3 was euthanized

prematurely 61 days pi due to a bacterial infection; PRNP 96 genotype was not determined.

b Two of 4 deer in cohort #4 (n=1 IC and n=1 PO) are alive and asymptomatic 24 months pi.

SOM References:

1. Spraker et al., Vet. Pathol. 39, 546 (2002).

2. E. S. Williams, S. Young, J. Wildl. Dis. 16, 89 (1980).

3. E. S. Williams, S. Young, J. Wildl. Dis. 18, 465 (1982).

4. M. W. Miller, E. S. Williams, Curr. Top. Microbiol. Immunol. 284, 193 (2004).
TSS



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