Bioassay studies of white-tailed deer.
White-tailed deer fawns were provided by the Warnell School of Forestry and Natural Resources, University of Georgia, Athens—a region in which CWD has not been detected. The deer fawns were hand raised and human and indoor adapted before overnight transport directly to the Colorado State University (CSU) CWD research indoor isolation facility without contact with the native Colorado environment. The 4-month-old fawns were adapted to the facility housing conditions and diet for 2 months before the study start.
All white-tailed deer were genotyped to determine their GG/GS (codon 96) status by the laboratory of Katherine O'Rourke, USDA-ARS, Pullman, WA. Deer were allocated into inoculation cohorts (n = 4) without knowledge of their G96 genotypes.
Protocols to preclude extraneous exposure and cross contamination between cohorts of animals as previously described (53
) incorporated protective shower-in requirements, Tyvek clothing, masks, head covers, and footwear while maintaining stringent husbandry. Tonsil biopsy and terminal sample collections were taken with animal-specific biopsy and sample collection instruments to minimize the possibility of cross contamination. Bedding and liquid waste from each suite were either incinerated or collected in a dedicated outdoor underground holding tank and denatured by alkaline digestion.
Deer inoculation cohorts.
Groups of 6-month-old fawns (usually four per group) (Table ) were housed in separate isolation suites throughout the study. Suite-dedicated protective clothing, utensils, and waste disposal were incorporated to exclude cross contamination by fomites, bedding, food, excretions, or contact. Deer cohorts 1 to 6 were inoculated by the intravenous (i.v.) route with blood components from CWD-infected donor deer housed in the CSU CWD isolation facility as follows: cohort 1, whole blood (250 ml); cohort 2, blood mononuclear cell fraction (1 × 107
to 1.24 × 108
white blood cells [WBC] plus platelets); cohort 3, cell-free plasma fraction (140 to 150 ml) recovered from 250 ml citrated whole blood; cohort 4, B cells (1 × 106
to 5 × 106
) separated by Dynatec magnetic bead separation (98% purity) using anti-sheep B-cell monoclonal antibody (MAb) 2-104, which identifies peripheral blood B cells and may identify follicular dendritic cells (FDCs) in lymphoid germinal centers (80
); cohort 5, CD41/61+
platelets (6 × 109
to 35 × 109
) magnetically separated to 99% purity using MAb CAPPA 2A (VMRD, Pullman, WA); cohort 6, CD14+
cells (4 × 105
) magnetically separated to 98% purity using anti-sheep CD14 MAb clone VPM65 (Fitzgerald Industries Inc., Concord, MA); cohort 7, naïve white-tailed deer inoculated with blood from CWD−
deer, which served as negative controls for the study and were housed in a separate suite at the same facility.
White-tailed deer cohorts i.v. inoculated with blood components
Blood donor deer.
Six experimentally inoculated CWD+
and two CWD−
deer housed at the CSU indoor research facility were recruited from previously described studies (54
) for use as blood donors for these studies (Table ).
CWD+/CWD− blood cell component donor history
Blood donors for cohorts 1, 2, and 3.
Two CWD-infected deer previously inoculated intracranially (i.c.) with 1 g whole brain homogenate collected from a naturally infected CWD+ deer (TS-989-09147) were the source animals.
Blood donors for cohorts 1, 4, 5, and 6.
Four CWD-infected deer served as blood donors for cohorts 1, 4, 5, and 6. Two donors (designated brain pool) had been orally inoculated with 10 g (2 g/day for 5 days) brain from naturally infected field isolates (TS-989-09147 or WDNR). The remaining two donors (designated blood pool) had received 250 ml of blood via i.v. infusion from an experimentally inoculated CWD+ deer (TS989-CSU112). Two deer from each cohort (1, 4, 5, or 6) were inoculated with whole blood or specific cell phenotypes from the brain pool donors, while the other two deer from each cohort were inoculated with similar components from the blood pool donors.
Blood donors for cohort 7.
One CWD− deer, housed at the Warnell School of Forestry and Natural Resources, University of Georgia, Athens (UGA)—a region where CWD has not been detected—served as the donor for two negative control donors that each received 250 ml whole blood via i.v. infusion.
Thus, the inocula used reflected both a conscious attempt to assess the universality of the results obtained given the constraints of a limited number of recipient animals and limited amounts of inoculum materials (cell fractions etc.) available.
Blood collections, harvests, and inoculations. (i) Cohorts 1 to 3.
One liter sodium citrate-treated whole blood was collected from each of two CWD+ donor deer (Table ) for cohort 1 to 3 inoculations (Table ). The blood was not pooled. Half (500 ml) of each whole blood collection was immediately administered i.v. to two recipient cohort 1 deer (250 ml/deer)/donor deer (four recipients). Plasma and blood mononuclear cells harvested from 250-ml nonpooled aliquots of whole blood were administered i.v. to cohorts 2 and 3 (Table ).
(ii) Cohorts 1, 4, 5, and 6.
Similarly, a portion (250 ml) of the whole blood collected from the blood pool and brain pool donors (Table ) was administered i.v. to two recipient cohort 1 deer/donor (Table ) (four recipients). The remaining blood collected from the blood pool was pooled, as was the remaining blood from the brain pool, and each pool was further processed to harvest specific cell phenotypes by magnetic separation (as described below) that were then inoculated by i.v. infusion into cohort 4, 5, or 6 (Table ).
Deer monitoring and sample collection.
All animals were monitored for evidence of CWD infection by serial tonsil biopsies taken at 3, 6, 12, and 15 months postinoculation (p.i.), and at study termination (19 months p.i.). Tonsil tissue was divided, and equal portions were either stored at −70°C or fixed in 10% formalin for 24 h before processing for immunohistochemistry (IHC) analysis. At the same sampling intervals, blood, saliva, feces, and urine were collected from each animal and stored at −70°C. At necropsy, the palatine tonsils, brainstem (medulla at the obex), and retropharyngeal lymph nodes, as well as other tissues, were collected for examination by IHC and Western blotting (WB) analyses to identify the presence of the protease-resistant prion protein associated with CWD (PrPCWD).
Cervid PrP transgenic mouse bioassay studies. (i) Cervid PrP transgenic mice.
), which express the elk PrP coding sequence, were generated in the Telling laboratory at the University of Kentucky. Mice were inoculated and maintained in accord with CSU IACUC guidelines.
All mice were screened at weaning for the presence of the cervid/elk Prnp transgene by both conventional and real-time PCR. All inoculated mice that tested negative for cervid PrPRES at the completion of bioassay studies were rescreened to confirm the presence of the cervid Prnp transgene.
The protocols for white-tailed deer described above also applied to cohorts of mice housed in filter-top isolation cages.
Mouse inoculation cohorts.
Groups of five to nine weanling mice (Table ) were housed in separate cages throughout the study. Suite-dedicated protective clothing, utensils, and waste disposal were incorporated to exclude cross contamination by fomites, bedding, food, excretions, or contact. Cohorts 8 and 9 consisted of naïve Tg(CerPrP-E226)5037+/− mice that served as i.c. brain inoculate positive or negative controls each receiving 30 μl of a 1% brain homogenate prepared in phosphate-buffered saline (PBS) of either CWD+ deer D10 or CWD− deer UGA. Cohorts 10 to 25 consisted of naïve Tg(CerPrP-E226)5037+/− mice that were inoculated by the i.c., i.v., intraperitoneal (i.p.), or per os (p.o.) route using the same CWD+ blood components described for white-tailed deer inoculations above. Cohorts 10 to 13 received whole blood. Cohorts 14 to 17 received the blood mononuclear cell fraction (106 WBC plus platelets). Cohorts 18 to 21 received the cell-free plasma fraction. Cohorts 22 and 23 received B cells (106) harvested from the retropharyngeal lymph node or spleen. Cohort 24 received CD41/61+ platelets (109). Cohort 25 received CD14+ cells (105). Cohorts 26 to 41, consisting of five to nine naïve Tg(CerPrP-E226)5037+/− mice, served as the negative controls for this study and were inoculated i.c., i.v., i.p., or p.o. with blood components from the same negative control white-tailed deer donors (Table ) as used for the negative control white-tailed deer inoculations (Table ).
Tg(CerPrP) mouse cohorts inoculated with blood components from CWD+ donor deer
Mouse monitoring and sample collection.
All mice were monitored daily for evidence of CWD clinical disease. Upon detection of clinical disease, mice were euthanized and necropsied. Brain tissue was collected, divided into equal portions, and either stored at −70°C for WB or fixed in 10% formalin for 24 h before processing for IHC analysis to identify the presence of PrPCWD.
Blood cell and plasma harvests [white-tailed deer and Tg(CerPrP-E226)5037+/− mouse inocula].
Total blood cell populations were collected from 250 ml sodium citrate-treated whole blood by centrifugation at 1,200 rpm for 15 min at 4°C. The plasma fraction was collected and set aside on ice. The cell fraction from this initial centrifugation was diluted 1:1 in 1× PBS (Gibco, Inc.) and layered over Histopaque 1088 (Sigma) at a 1:1 ratio. These Histopaque gradients were centrifuged without a brake at 2,500 rpm for 30 min at room temperature. The discrete bands of WBC were collected, diluted in an equal volume of 1× PBS, and further centrifuged for 10 min at 2,500 rpm at 4°C (washed). The cell pellets were washed in wash buffer (1× PBS, 0.2% fetal bovine serum [FBS], 2 mM EDTA) three times. Platelets were collected from the plasma fraction by centrifugation at 3,000 rpm for 15 min. Cells and plasma recovered from Histopaque 1088 gradient separations and plasma centrifugations were either directly inoculated into deer and mouse bioassay studies or further processed to separate cell phenotypes.
Retropharyngeal lymph node and spleen cell harvests [Tg(CerPrP-E226)5037+/− mouse inocula].
Retropharyngeal lymph node and spleen tissues were pressed through a fine wire mesh (0.45 μm), and WBC were collected as described above for the Histopaque gradient protocol.
Cell phenotype labeling and flow cytometry.
Cell phenotype MAbs were used to recover and determine the purity of 2-104+
B cells, CD14+
monocytes, and CD41/61+
platelets from the blood donor sources described above. Leukocyte and platelet blood cells were collected by centrifugation and Histopaque 1088 separation as described above and were then labeled with one of three antibodies (Table ), i.e., anti-sheep pan-B-cell MAb 2-104 (equivalent to MAb 2-8 described by Young et al. [80
]) (cell supernatant used undiluted), anti-sheep CD14 MAb clone VPM65 (cell supernatant used undiluted; Fitzgerald Industries Inc., Concord, MA), or anti-sheep CD41/61 MAb CAPPA 2A (1:100 dilution of a 1-mg/ml stock; VMRD, Pullman, WA). Cell aliquots were incubated with primary antibody for 20 min on ice and then washed three times in wash buffer. The secondary antibody, goat anti-mouse IgG or IgM fluorescein isothiocyanate (FITC), was diluted 1:100 in 1× PBS, 0.2% FBS and placed on the cells for 20 min on ice. The cells were again washed three times in wash buffer. The cells were then labeled with anti-FITC beads at 10 μl beads/107
cells, again incubated on ice for 20 min, and passed over LS or LD Dynatec magnetic bead separation columns (in accordance with the manufacturer's instructions). Cell populations of interest were eluted in 1× PBS containing 0.1% FBS. Eluted (FITC-labeled) cells were analyzed by flow cytometry (Dako-Becton Dickinson). To determine purity, the cells were gated by forward and side scatter to include primarily lymphocytes, which were counted, and volumes were adjusted to be equal to or greater than the total number of each cell-specific phenotype populating 1 × 107
peripheral blood mononuclear cells (as determined by prior flow cytometric analysis of specific cell phenotype populations in white-tailed deer; i.e., 2-104+
B cell populations were ~10% and CD14+
cells ~2% of the total leukocyte population). This was done to equate the total number of phenotype-specific cells (2-104+
) to that found in the total blood mononuclear cell fraction inoculum (cohort 2) that established infection (107
blood mononuclear cells) (Tables and ). The cells were either directly inoculated by i.v. inoculation into deer bioassays or frozen for future i.c. mouse bioassays.
MAbs used for cell-specific phenotype harvests
Bioassay results from naïve deer cohorts inoculated with CWD+ blood components
Tissue homogenates were prepared from the obex region of the medulla oblongata encompassing the dorsal motor vagal nucleus (medulla at the obex). Ten percent (wt/vol) homogenates were prepared in NP-40 buffer (10 mM Tris-HCl buffer [pH 7.5], 0.5% NP-40, 0.5% sodium deoxycholate) by Fastprep disruption at a setting of 6.5 for 45 s. Twenty-five microliters of each homogenate was mixed with 5 μl of proteinase K (PK; Invitrogen) to a final concentration of 20 μg/ml and incubated for 30 min at 37°C with shaking. PK activity was stopped with 4 μl 200 mM Pefablock SC, and an equivalent volume of each sample was mixed with 10 μl sample buffer (20% 10× reducing agent, 50% 4× LDS sample buffer; Invitrogen) and 5 μl NP-40 buffer (10 mM Tris-HCl [pH 7.5], 0.5% deoxycholic acid, 0.5% nonylphenoxylpolyethoxylethanol), heated to 95°C for 5 min, and separated by 12% Bis-Tris precast polyacrylamide gel electrophoresis (PAGE) (Invitrogen) at 150 V for 2.5 h in 1× morpholinepropanesulfonic acid (MOPS; Invitrogen). Proteins separated by PAGE were transferred to polyvinylidene fluoride (PVDF) membrane for 1 h at 100 V in transfer buffer (0.025 M Trizma base, 0.2 M glycine, 20% methanol, pH 8.3). After the PVDF membranes were blocked overnight at room temperature in Pierce Blocker, they were probed with PrP-specific antibody BAR224 (kindly supplied by J. Grassi), followed by horseradish peroxidase-conjugated goat anti-mouse IgG diluted in Pierce Blocker. Membranes were washed for 1 h after blocking and between antibodies with wash buffer (0.1 M Tris, 0.15 M NaCl, 0.2% Tween 20, pH 8.0). To visualize PrP bands, the PVDF membranes were developed with the Amersham ECL detection system and a digital GelDoc (Fuji Intelligent dark box) using LAS-3000 Lite ImageReader software.
IHC analysis was performed by employing protocols described by Spraker et al. (69
). Briefly, 3- to 5-mm sections of formalin-fixed, formic acid-treated tissues were deparaffinized at 60 to 70°C for 1 h, rehydrated via a series of xylene-ethanol baths, and treated in formic acid a second time (5 min) prior to a 20-min antigen retrieval (10× Dako target retrieval solution) cycle in a 2100 Retriever (PickCell Laboratories). Slides were further processed with the aid of a Ventana Discovery autostainer utilizing the Ventana Red Map stain kit, PrPCWD
-specific primary antibody BAR224, and a biotinylated secondary goat anti-mouse antibody (Ventana). After autostaining, the slides were quickly rinsed in a warm water detergent solution, passed through a series of dehydration baths, and coverslipped.