Salivary prions in sheep and deer

Prion 6:1, 52-61; January/February/March 2012; © 2012 Landes Bioscience

RESEARCH PAPER

52 Prion Volume 6 Issue 1

*Correspondence to: Stanley B. Prusiner; Email: [email protected]: 06/17/11; Revised: 07/28/11; Accepted: 07/29/11http://dx.doi.org/10.4161/pri.6.1.16984

Introduction

Scrapie of sheep and chronic wasting disease (CWD) of cervids are highly transmissible, fatal prion diseases that cause neuro-degeneration.1,2 Epidemiologic and experimental data argue that scrapie and CWD can be transmitted horizontally.2-5 Sheep and cervids can be infected orally6,7 and seem to be able to contract prions from contaminated environments where prions can per-sist for long periods of time.8,9 In addition to being found in the central nervous system (CNS) and lymphoreticular system, CWD prions have been identified in muscle, blood and other tissues. Moreover, cervids with clinical signs of CWD have been shown to shed prions in feces, saliva and urine, which may contribute to the horizontal spread.10-12 Transmission experi-ments of γ-irradiated fecal samples from CWD-infected mule deer to transgenic (Tg) mice expressing elk prion protein (PrP), denoted Tg(ElkPrP) mice, have shown that mule deer infected with CWD prions start shedding prions in feces at 9 mo post-infection, long before they show clinical symptoms.12 Oral trans-mission experiments to mule deer with saliva collected 6–13 mo post-oral infection from presymptomatic mule deer have shown that presymptomatic shedding of CWD prions also occurs in

Scrapie of sheep and chronic wasting disease (CWD) of cervids are transmissible prion diseases. Milk and placenta have been identified as sources of scrapie prions but do not explain horizontal transmission. In contrast, CWD prions have been reported in saliva, urine and feces, which are thought to be responsible for horizontal transmission. While the titers of CWD prions have been measured in feces, levels in saliva or urine are unknown. Because sheep produce ~17 L/day of saliva and scrapie prions are present in tongue and salivary glands of infected sheep, we asked if scrapie prions are shed in saliva. We inoculated transgenic (Tg) mice expressing ovine prion protein, Tg(OvPrP) mice, with saliva from seven Cheviot sheep with scrapie. Six of seven samples transmitted prions to Tg(OvPrP) mice with titers of -0.5 to 1.7 log ID50 U/ml. Similarly, inoculation of saliva samples from two mule deer with CWD transmitted prions to Tg(ElkPrP) mice with titers of -1.1 to -0.4 log ID50 U/ml. Assuming similar shedding kinetics for salivary prions as those for fecal prions of deer, we estimated the secreted salivary prion dose over a 10-mo period to be as high as 8.4 log ID50 units for sheep and 7.0 log ID50 units for deer. These estimates are similar to 7.9 log ID50 units of fecal CWD prions for deer. Because saliva is mostly swallowed, salivary prions may reinfect tissues of the gastrointestinal tract and contribute to fecal prion shedding. Salivary prions shed into the environment provide an additional mechanism for horizontal prion transmission.

Salivary prions in sheep and deerGültekin Tamgüney,1,2,† Jürgen A. Richt,3,8 Amir N. Hamir,3,9 Justin J. Greenlee,3 Michael W. Miller,4 Lisa L. Wolfe,4

Tracey M. Sirochman,4 Alan J. Young,5 David V. Glidden,6 Natrina L. Johnson,1 Kurt Giles,1,2 Stephen J. DeArmond1,7 and Stanley B. Prusiner1,2,*

1Institute for Neurodegenerative Diseases; San Francisco, CA USA; 2Department of Neurology; University of California, San Francisco, CA USA; 3National Animal Disease Center; ARS-USDA; Ames, IA USA; 4Colorado Division of Wildlife; Wildlife Research Center; Fort Collins, CO USA; 5Department of Veterinary Science; South Dakota State University;

Brookings, SD USA; 6Departments of Epidemiology and Biostatistics; University of California, San Francisco, CA USA; 7Department of Pathology; University of California; San Francisco, CA USA; 8College of Veterinary Medicine; Kansas State University, Manhattan, KS USA; 9MD Anderson Cancer Center; Houston, TX USA

†Current address: German Center for Neurodegenerative Diseases; Bonn, Germany

Key words: scrapie, chronic wasting disease, saliva, horizontal transmission, titers

saliva.13 Whether presymptomatic prion shedding occurs in urine of CWD-infected deer remains to be determined.

In contrast to CWD prions, how scrapie prions are shed into the environment remains unclear. Previous studies identified scrapie prions in various non-CNS tissues including tonsils, ret-ropharyngeal and mesenteric-portal lymph nodes, spleen, Peyer’s patches, placenta, blood, tongue and salivary glands.3,14-17 Prions were found in mammary glands of sheep harboring scrapie prions and maedi-visna viruses (MVV),18 and in the milk of asymp-tomatic, scrapie-infected ewes.19-21 Although prion-infected milk may contribute to the spread of scrapie,19,22 it does not explain the observed patterns of natural scrapie. Despite the similar tissue distribution of prions in sheep with scrapie and cervids with CWD, scrapie prions have not been reported in saliva, urine or feces. Because sheep secrete large volumes of saliva (~17 L/day)23,24 and prions have been identified in the tongue and salivary glands of sheep with scrapie, we investigated whether pri-ons are secreted into the saliva. We collected and concentrated saliva samples from seven scrapie-infected Cheviot sheep, then intracerebrally (i.c.) inoculated the samples into Tg mice express-ing ovine prion protein, denoted Tg(OvPrP) mice. Comparison of the resulting incubation times with those from endpoint titra-tions of scrapie brain homogenates in Tg(OvPrP) mice enabled us

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saliva samples and then humanely euthanized the sheep. Scrapie infection was confirmed in each sheep by spongiform changes in the CNS and the presence of PrPSc by immunohistochemistry (Table 2 and Fig. S1). The saliva samples, 1.26 ml each, were treated with sodium phosphotungstate to precipitate selectively PrPSc present in the samples;25 concentrated saliva samples were then bioassayed by i.c. inoculation into Tg(OvPrP) mice.26 These Tg(OvPrP+/+) mice were homozygous for the transgene encoding ovine PrP with the VRQ polymorphism.

Six of the seven saliva concentrates from sheep with clini-cal scrapie transmitted prion disease to Tg(OvPrP) mice with median incubation times ranging from 125 to 184 dpi (Table 1 and Fig. 1A). Overall, 21 of the 43 (49%) inoculated Tg mice developed clinical signs of neurologic disease within observation periods of over 500 d. In contrast, Tg(OvPrP) mice i.c. inocu-lated with saliva samples collected from 4 uninoculated, healthy Cheviot sheep remained free of neurologic signs (Table 1). Biochemical analysis of brains from diseased Tg(OvPrP) mice revealed the presence of PrPSc upon limited digestion with protein-ase K (PK) in protein gel blots (Figs. 1B and S2). Neuropathologic analysis of these brains also showed the presence of spongiform degeneration, accompanied by PrPSc deposition and intense astro-cytic gliosis (Fig. 2), similar to the neuropathology observed in Tg(OvPrP) mice infected with scrapie prions derived from sheep brain tissue.26

To estimate the infectivity of tissues and secretions contain-ing scrapie prions, we performed endpoint titrations with sheep scrapie strain SSBP/1 prions by i.c. inoculation into Tg(OvPrP) mice using ten 10-fold dilutions of a 10% brain homogenate ranging from 10-1 to 10-10 (Fig. 3). Based on Cox regression anal-ysis, which estimates the infectious dose (ID

50) based on Kaplan-

Meier survival times and accounts for censored events,12 the titer of the 10% SSBP/1 scrapie brain homogenate was 7.2 log ID

50 U/

ml with a 95% confidence interval (CI) between 6.8 and 7.7 log

to determine the prion titer in saliva. Transmission experiments to Tg(ElkPrP) mice with concentrated saliva samples collected from two mule deer with clinical signs of CWD produced simi-lar findings: CWD and scrapie prions were shed in saliva of deer and sheep, respectively. Considering that sheep produce large volumes of saliva daily and that most of the saliva is swallowed, salivary prions could contribute to environmental contamination either directly or through the feces. Our observations raise the possibility that swallowed salivary prions might feature in the horizontal transmission of scrapie and CWD.

Results

To assess prion secretion in saliva, we i.c. inoculated scrapie prions into seven Cheviot sheep expressing PrP with the VRQ/ARQ polymorphisms at codons 136, 154 and 171, respectively (Table 1). When these sheep developed clinical signs of scrapie in between 171 and 250 d postinoculation (dpi), we collected

Table 1. Transmission of prions in sheep saliva to Tg(OvPrP) mice

Inoculum (Sheep ID) Scrapie onset (days) Sheep PrP genotype|| Median incubation days (95% CI*) n/n0§ Log dose ID50 U/ml

(95% CI*)‡

444 188 VRQ/ARQ 125 (118, 139) 8/8 1.7 (0.9, 2.4)

429 171 VRQ/ARQ 139 (133, 230) 5/7 1.1 (0.2, 2.0)

430 171 VRQ/ARQ 131 (113, 158) 3/7 0.8 (-0.4, 2.0)

438 201 VRQ/ARQ 157 (148, 165) 2/5 0.6 (-0.8, 2.0)

419 250 VRQ/ARQ 184 (119, 249) 2/6 0.3 (-1.1, -1.7)

437 239 VRQ/ARQ 139 1/5 -0.5 (-2.4, 1.5)

427 250 VRQ/ARQ >500 0/5 -

R8† >700 VRQ/VRQ >500 0/5 -

R10† >700 VRQ/VRQ >500 0/5 -

365† >700 VRQ/ARQ >500 0/6 -

370† >700 VRQ/ARQ >500 0/6 -

||Residues at polymorphic positions 136, 154 and 171. *CI, confidence interval. §n, number of ill animals; n0, number of inoculated animals. ‡Log dose in ID50 U/ml represents the equivalent titer of 1 ml of unconcentrated saliva relative to 30 µl of a 10% SSBP/1 brain homogenate. †Saliva samples were collected at ~700 d from healthy, uninoculated sheep.

Table 2. Neuropathology results for scrapie-infected sheep saliva donorsa

Sheep ID#

H&E-based diagnosis of spongiform encephalopathy

IHC for PrPSc

IHC for GFAPb

444 +++ ++++ +

429 +++ ++++ +

430 +++ ++++ +

438 ++ ++++ +

419 ++ ++++ +437 ++ ++++ +427 ++ ++++ +

aFor corresponding scores, see Material and Methods. bIHC for GFAP indicates reactive astrocytic gliosis; relative scores could not be assigned because there was not a wide range of intensities.


Salivary prions that pass through the GI tract may also contrib-ute to the spread of prions in feces (Table 4).12,30 Our findings also indicate that scrapie prions can be shed into the environment in saliva from sheep with clinical scrapie. Thus, contamination of shared water and feed sources, pasture vegetation and soil with prion-containing saliva may contribute to the horizontal trans-mission of scrapie among sheep and possibly goats.2,3 Because prion titers in saliva were relatively low, frequent exposure of sheep to saliva-contaminated water, feed and environments may increase their overall probability of infection.31 Although we only measured prions in saliva of sheep with clinical signs of disease, scrapie-infected sheep may shed prions in saliva before they become symptomatic, as observed for CWD prions in saliva of deer.13 Prions in saliva may originate from salivary glands or tongue that have been shown to contain prions in infected sheep

ID50

U/ml. Thus, an entire sheep scrapie brain weighing 109 g contains 10.2 log ID

50 U (Eq. S1).27 Based on Cox

regression analysis, the equivalent titers of scrapie prions in the tested saliva samples were between -0.5 to 1.7 log ID

50

U/ml (Table 1). Considering that an adult ewe weighing 71.2 kg produces 16.6 L of saliva per day,23,24 and assum-ing constant salivary shedding of scrapie prions in the late stages of disease, we estimate that the titers of scrapie prions in saliva were 6.2–8.4 log ID

50 units over a 10-mo

period (Eq. S2). This number of ovine prions in saliva is similar to that calculated for CWD prions in feces over the same time period.28

To measure prions in saliva excreted from deer with CWD, we orally inoculated CWD prions into 2 mule deer expressing PrP with the QGAS/QGAS polymorphisms at codons 95, 96, 116 and 225, respectively (Table 3). When these deer developed clinical signs of CWD at 427 and 658 dpi, we collected saliva samples (5 ml each) and then euthanized the deer. CWD was confirmed in both mule deer at 251 dpi by positive tonsil and rectal mucosa biop-sies.29 Similar to the treatment of the sheep saliva samples, the prions in these saliva samples were precipitated with sodium phosphotungstate and then bioassayed by i.c. inoc-ulation into Tg(ElkPrP) mice.26 These Tg(ElkPrP+/+) mice are homozygous for the transgene that expresses ElkPrP with the M132 polymorphism.

Both saliva concentrates from deer with clinical CWD transmitted prion disease to Tg(ElkPrP) mice. One sample transmitted to 4 of 4 mice with a median incubation time of 287 dpi, whereas the other saliva sample only transmit-ted to 1 of 7 mice at 438 dpi (Table 3 and Fig. 4A). In contrast, Tg(ElkPrP) mice infected with a saliva sample collected from an uninoculated, healthy deer remained free of neurologic signs (Table 3). Biochemical analysis of brains from diseased Tg(ElkPrP) mice revealed PrPSc upon limited PK digestion (Fig. 4B). Neuropathology showed spongiform degeneration, PrPSc deposition and an intense astrocytic gliosis (Fig. 5).

Based on endpoint titration results that we had obtained with the Elk1 CWD isolate inoculated into Tg(ElkPrP) mice,12 we estimated the titer in the deer saliva samples using Cox regression analysis. The titers of CWD prions in the saliva samples were between -1.1 and 0.4 log ID

50 U/ml (Table 3).

An adult female mule deer weighing 65 kg produces 13.5 L saliva per day.24 Assuming that salivary prions may be shed as early as fecal prions in mule deer, we estimated the titer of CWD prions in saliva over a 10-mo period to be between 5.5 and 7.0 log ID

50

U (Eq. S3).

Discussion

We conclude that sheep with clinical scrapie can shed infectious prions in saliva. Because saliva is mostly swallowed, salivary pri-ons may be a continuous source for primary or secondary infec-tion of lymphoid, epithelial, and other susceptible tissues of the GI tract and may thus contribute to peripheral prion replication.

Figure 1. (A) Kaplan-Meier plots indicating incubation times in Tg(OvPrP) mice after i.c. inoculation with concentrated saliva samples collected from sheep (ID numbers indicated) once they developed scrapie in 171–250 d after i.c. infec-tion with scrapie prions. Six of seven saliva samples transmitted prion disease to Tg(OvPrP) mice. (B) Protein gel blots of brain homogenates of Tg(OvPrP) mice after i.c. inoculation with either saliva samples collected from scrapie sick sheep (ID numbers indicated; lanes 1–6) or with SSBP/1 scrapie brain homog-enate (lanes 9 and 10). Brain homogenates of uninoculated Tg(OvPrP) mice are shown as controls (lanes 7 and 8). Samples were undigested (-) or digested with proteinase K (+). Molecular masses of protein standards are shown in kilodal-tons (kDa).


and deer.15,16,32 Alternatively, prions may be shed into saliva from tissues associated with the lymphoreticular system of the upper alimentary tract, such as the ton-sils or the lymph nodes associated with the naso-oro-pharyngeal cavity.3,33 Experiments in prion-infected rodents have shown that the oral and nasal mucosa, including the papillae in the tongue, can harbor pri-ons and may act as potential sources for the horizon-tal transmission of animal prion diseases since these tissues may release prions during their normal turn-over.34,35 Equally, the tongue, and the oral and nasal mucosa can also act as routes for neuroinvasion.36-39

Although prions were reported previously in the saliva of CWD-infected mule deer,10 the titers were not determined. A pooled and concentrated (10-fold) saliva sample from five white-tailed deer with CWD was reported to transmit prion disease to 8 of 9 Tg(CerPrP+/-)1536 mice in 342 ± 102 dpi.11 Based on published titration results with a pooled elk CWD inoculum in Tg(CerPrP+/-)1536 mice,40 we estimated the equivalent titer for this pooled CWD saliva sample to be -0.7 log ID

50 U, which is similar to our value of

Figure 2. Neuropathology of brain sections from Tg(OvPrP) mice inoculated with saliva from scrapie-sick sheep (G–I) or with brain homogenate from scrapie-sick sheep that had been inoculated with SSBP/1 prions (D–F). Brain sections from uninoculated, 612-d-old, control Tg(OvPrP) mice are shown as controls (A–C). Sections show vacuolation (left column), astrocytic gliosis (middle column) and PrPSc deposition (right column). Tg(OvPrP) mice inoculated with saliva samples showed vacuolation (G), severe gliosis (H) and PrPSc deposits (I), similar to Tg(OvPrP) mice inoculated with scrapie-in-fected, sheep brain homogenate (D–F). Control mice showed no vacuolation (A), no gliosis (B) and no PrPSc deposits (C). Bar in I represents 50 μm and applies to all parts.

Figure 3. Endpoint titration of SSBP/1 prions in Tg(OvPrP) mice. Ten dilutions, rang-ing from 10-1 to 10-10 as indicated, of a 10% (wt/vol) brain homogenate were i.c. in-oculated into 30 groups of 4 mice each. The percentage of mice without neurologic disease is plotted against the incubation time, in days post-inoculation.


Animal Care and Use Committee, under the CAR “Incubation periods of prion diseases” (AN075039-03). The sheep experi-ments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Washington, DC) and the Guide for the Care and Use of Agricultural Animals in Research and Teaching (Federation of Animal Science Societies, Champaign, IL); the protocol was approved by the Institutional Animal Care and Use Committee at the National Animal

-0.9 log ID50

U for scrapie saliva sample 444 based on 30 μl of 10% saliva preparations (Table 1).

A recent study using serial protein misfolding cyclic amplification (sPMCA) reported the pres-ence of scrapie prions in buccal swab samples of pre-symptomatic and symptomatic sheep with scrapie.41 In contrast to bioassays, sPMCA is non-quantitative and can generate spontaneous false-positive results, which can be especially problematic when prion titers are low and multiple amplification cycles are used for detection.41,42

Our results indicate a similar magnitude of salivary prion shedding between sheep and deer. Although salivary prion titers are much lower in comparison to those measured in brain, spleen and lymph nodes of affected animals, the num-ber of prions secreted in saliva over the incubation period may approach that found in the brains of terminally ill animals, assuming that, similar to fecal prion shedding, salivary prion shedding is not restricted to terminally sick animals (Table 4). Also, possible differences between prion strains and animal species used in titration studies may affect the estimated titers. Whether these assumptions are accurate requires additional studies. It remains to be determined at what quantities scrapie prions are shed in urine and feces of scrapie-infected sheep (Table 4) as well as when and to what level this shedding may occur in presymptomatic animals. Whether prions shed in saliva have different strain characteristics from prions shed in feces is also unknown. In light of the similarities in peripheral prion distribution patterns and shedding of infec-tious prions by small ruminants and cervids, it will be important to determine whether patients with variant Creutzfeldt-Jakob disease (vCJD) who har-bor prions in their lymphoreticular tissues,43,44 also shed vCJD prions in saliva, urine or feces.

Materials and Methods

Ethics statement. All mouse studies were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health; protocols were reviewed and approved by the UCSF Institutional

Table 3. Transmission of prions in deer saliva to Tg(ElkPrP) micea

Inoculum (Deer ID¶) CWD onset (days) Median incubation time in days (95% CI*) n/n0† Log dose ID50 U/ml (95% CI*)‡

36A04 658 287 (287, 491) 4/4 0.4 (-0.5, 1.3)

D604 427|| 438 1/7 -1.1 (-2.9, 0.7)

SA** >800 >607 0/7 -aAll inoculated deer expressed QGAS/QGAS at PrP codons 95, 96, 116 and 225, respectively. All deer tested positive for prion disease upon tonsil and rectal mucosa biopsy at 251 dpi. ¶Saliva samples collected from three additional deer with CWD caused intercurrent disease in all inoculated mice. *CI, confidence interval. †n, number of ill animals; n0, number of inoculated animals. ‡Log dose in ID50 U/ml represents the equivalent titer of 1 ml of uncon-centrated saliva relative to 30 µl of a 10% Elk1 brain homogenate. ||This animal was euthanized because of a brain abcess. **Saliva sample was collected from this healthy, uninoculated white-tailed deer at 800 d.

Figure 4. (A) Kaplan-Meier plots showing incubation times in Tg(ElkPrP) mice after i.c. inoculation with concentrated saliva samples collected from two mule deer (D604 and 36A04) at 427 and 658 d, respectively, after oral infection with CWD prions. (B) Protein gel blots of brain homogenates of Tg(ElkPrP) mice that were inoculated i.c. either with saliva samples collected from mule deer with CWD (ID numbers indicated; lanes 1–10) or with CWD brain homogenate from mule deer G1204 (lanes 13 and 14). Brain homogenates from uninoculated Tg(ElkPrP) mice are shown as controls (lanes 11 and 12). Samples were undigested (-) or digested with proteinase K (+). Molecular masses of protein standards are shown in kilodaltons (kDa).


Housing, care and inoculation of sheep. Seven 4-mo-old Cheviot lambs from a scrapie-free flock were obtained for this study and trans-ported to the NADC (Ames, IA) where they were inoculated with scrapie prions. For negative con-trols, four uninoculated Cheviot sheep from the sheep flock at South Dakota State University were selected.

For the scrapie inoculation, No. x124 was prepared from a pool of seven sheep brains from five flocks that were scrapie-positive by immu-nohistochemical and protein gel blot analyses.47 The infected brains were homogenized by ultrasoni-cation at a final concentration of 20% (wt/vol) in phosphate-buff-ered saline (PBS). This stock was diluted to 10% (wt/vol) in PBS at the time of inoculation.

The seven Cheviot lambs were inoculated i.c. with 1.0 ml (#430), 0.5 ml (#429), 0.2 ml (#419) or 0.1 ml (#437, 438, 427 and 444) of the No. x124 inoculum, using a proce-dure described previously in refer-ence 48. Briefly, the animals were

sedated with xylazine, a midline incision was made in the skin at the junction of the parietal and frontal bones, and a 1-mm hole was trephined through the calvarium. The inoculum was injected into the midbrain using a 22-gauge, 9-cm-long needle while withdrawing the needle from the brain. The skin incision was closed with a single suture. Inoculated animals were initially housed in a biosafety level-2 containment facility (2 per pen) and later moved to outside pens at the NADC. The sheep were fed pelleted growth and maintenance rations that contained no ruminant protein, and clean water was available ad libitum.

The scrapie-infected sheep were euthanized when they devel-oped terminal clinical signs of scrapie as determined by the attending veterinarian. They were examined at necropsy and tis-sue samples were collected.

Housing, care and inoculation of mule deer. Captive mule deer were held at the Colorado Division of Wildlife’s (CDOW) Foothills Wildlife Research Facility as part of an ongoing study on prion shedding patterns in North American cervid spe-cies.29,49 Mule deer fawns were acquired and bottle-raised using canned evaporated bovine milk and established protocols. Deer were confined to 0.1 ha, biosecure paddocks throughout the study, except when held in metabolic cages for sample collec-tions. Alfalfa hay, pelleted supplement (Ranch-Way deer diet and Mazuri Inc., “browser” ration), mineralized salt blocks and water were provided ad libitum in all paddocks as per standard feeding and husbandry protocols.

Disease Center (NADC #3805: “Sheep scrapie-minimum infec-tious dose”). Mule deer care and research protocols were per-formed in strict accordance with the rules and regulations of the United States Animal Welfare Act, reviewed and approved by the CDOW Animal Care and Use Committee (07-2004).

Mice and transmission studies. Production of Tg(OvPrP+/+)14882 and Tg(ElkPrP+/+)12584 mice has been described previously in reference 26. Tg(OvPrP) and Tg(ElkPrP) mice do not express endogenous mouse PrP and homozygously express the PRNP allele encoding OvPrP(V136, R154, Q171) and ElkPrP(M132), respectively, from the cosSHa.Tet cosmid vector.

The experimental SSBP/1 sheep scrapie inoculum was obtained from Nora Hunter at the Roslin Institute, University of Edinburgh (Edinburgh, UK). This inoculum was derived from the brain homogenates of three sheep with scrapie and then pas-saged mainly through NPU Cheviot sheep.26,45,46

Weanling mice were inoculated into the right parietal lobe of the cerebrum with 30 μl of sample using a 27-gauge, disposable hypodermic syringe. Inoculated mice were examined daily for their clinical status and thrice weekly for neurologic dysfunction and scored for prion disease based on established diagnostic crite-ria. Sick mice were euthanized and their brains collected. Half of the brain was frozen and the other half immersion-fixed in 10% neutrally buffered formalin for biochemical and neuropathologic analyses, respectively.

Figure 5. Neuropathology of brain sections from Tg(ElkPrP) mice inoculated with saliva (G–I) or with brain homogenate (D–F) from mule deer with CWD. Brain sections from uninoculated, 658-d-old, control Tg(ElkPrP) mice are shown as controls (A–C). Sections show vacuolation (left column), astrocytic gliosis (middle column), and PrPSc deposition (right column). Some Tg(ElkPrP) mice inoculated with saliva from CWD-positive mule deer developed neurologic symptoms, showed vacuolation (G), severe gliosis (H) and PrPSc deposits (I). Mice inoculated with brain homogenate from mule deer with CWD showed vacuolation (D), reactive astrocytic gliosis (E) and PrPSc deposits (F). Control mice showed no vacuolation (A), no gliosis (B) and no PrPSc deposits (C). Bar in I represents 50 μm and applies to all parts.


6.5 ml because the pellet was resuspended in 300 μl diluent con-taining 5% (wt/vol) bovine albumin Fraction V.

Statistical analysis of prion infectivity in Tg mice. The Cox proportional hazards model was used to calculate the ID

50 value

of a 10% SSBP/1 sheep scrapie brain homogenate as previously described for a 10% brain homogenate from an elk with CWD, denoted the Elk1 isolate.12,52 To calibrate titers in saliva samples from sheep and mule deer to an equivalent titer of a 10% brain homogenate of SSBP/1 or Elk1, we used a recently established approach.12 To compare onset times, we used statistical methods of survival analysis that can handle observations that are “cen-sored,” such as animals that die from competing, unrelated causes or that do not become ill during the study period.53 This method is advantageous when attempting to calibrate an inoculum that does not result in illness in all animals. The methods operate by considering k = 1,..., K different experiments to be compared with the serial dilution series of SSBP/1. Those experiments are then paired with the serial SSBP/1 dilution data. For the ith com-parison, the variable group

k is 1 for animals in the kth experiment,

0 for animals in the SSBP/1 serial dilution series, and dilutionk

gives the dilution in the kth experiment relative to a 10% homog-enate. The nonlinear regression model for the mean onset time in the kth series, denoted t

k, is log(t

k) = α

0 + α

1 group

k + α

2 dilution

k.

The equivalent log titer for the kth experiment series relative to the ID

50 of the SSBP/1 inoculum is then α

1 α

2 - log

10(ID

50 SSBP/1).

For cases in which not all animals die, the mean illness time cannot be defined, but the hazard function, which describes the rate of the onset of disease, can be defined. We used the Cox pro-portional hazards model,52 to represent the hazard as h

k(t) = h

0(t)

exp(β1 group

k + β

2 dilution

k). The equivalent log titer for the kth

At weaning, mule deer fawns were orally infected with approx-imately 1 g of pooled, CWD infectious brain material placed at the base of the tongue; based on previous analyses, the inoculum pool contained approximately 3 μg PrPSc per g of brain tissue50 and showed prion conversion in vitro and infectivity in vivo.7,50,51 Uninoculated, negative control deer were sampled under similar captive conditions, but were held in a different facility outside a geographic area where CWD is known to occur. All infected deer surviving >250 dpi showed evidence of PrPSc accumulation in tonsil and rectal mucosa biopsies,29 indicating successful infec-tion. All infected deer that survived >427 dpi showed clinical signs of CWD prior to death and evidence of prion infection on postmortem examination.

Saliva sampling and treatment. Saliva samples were col-lected from sheep with clinical signs of scrapie and from mule deer infected with CWD prions at the time of euthanasia as well as from age-matched, uninoculated and healthy control animals (Tables 1 and 2). The collected saliva samples were kept frozen in plastic tubes at -80°C until processed. To concentrate the scra-pie prions in the samples, 1.26 ml of sheep saliva was incubated overnight at 1,200 rpm and 37°C in the presence 1% (wt/vol) phosphotungstic acid (Sigma) at pH 7.4, 2x complete protease inhibitor cocktail mix (Roche Applied Science) and 2% (wt/vol) sarkosyl in a total volume of 1.5 ml.25 After a 30 min spin at 14,000x g at room temperature, the pellet was resuspended in 300 μl diluent containing 5% (wt/vol) bovine albumin Fraction V (ICN Biomedicals). Because sodium phosphotungstate pre-cipitates contain about 99% prions, this procedure concentrated prions ~4.2-fold.25 Similarly, CWD prions in deer saliva were concentrated 16.7-fold from 5 ml of saliva in a total volume of

Table 4. Prion titers in tissues and secretions of clinically sick sheep and deer†

Species Tissue Log ID50 U/ml wt or vol Total log ID50 units Reference

Sheep Brain 7.2a 109 g 10.2 this paper

Spleen 4.0a 30 mg 3.5 3

Retropharyngeal lymph node 3.8a 30 mg 3.3 3

Saliva 1.7b 4,980 L 8.4 this paper

Urine ND ND ND -

Feces ND ND ND -

Blood +c ND ND 17

Deer Brain 6.0a 200 g 9.3 12

Spleen +d ND ND 51

Retropharyngeal lymph node +e ND ND 56

Saliva 0.4b 4,050 L 7.0 this paper

Urine +f ND ND 11

Feces 1.5a,b 234 kg 7.9 12

Blood +g ND ND 57aPrion titers were determined in 10% tissue or fecal homogenate. bFor better comparison, salivary and fecal prion doses were both estimated for a dis-ease course of 10 mo. Salivary prion titers were determined from undiluted saliva. cScrapie was transmitted from donor sheep with preclinical scrapie to recipient sheep by transfusion of 450–500 ml whole blood as early as 575 d postinoculation. dCWD prions were detected by IHC as early as 189 d after oral inoculation of mule deer with brain tissue from CWD-infected mule deer. eFour of six Tg(CerPrP+/-)1536 mice developed neurologic symptoms in 515 ± 27 d after i.c. inoculation with 30 μl of a 1% tissue homogenate from a CWD-positive elk. fTwo of nine Tg(CerPrP+/-)1536 mice developed neuro-logic symptoms in 373 ± 3 d after i.c. inoculation with 30 μl of 10-fold-concentrated urine collected from 5 CWD-positive white-tailed deer. gSeven of nine Tg(CerPrP-E226+/-)5037 mice developed neurologic symptoms in 270 ± 5 d after i.c. inoculation with 30 μl of whole blood collected from a CWD-positive white-tailed deer. ND, Not determined.


(Dako North America, Inc.) containing all reagents except for primary antibody. Blocking solution, provided in the kit, was applied for 5 min. The slides were incubated with a polyclonal rabbit anti (Dako North America, Inc.,) at 1:15,000 dilution for 3 h at room temperature. Polymer/HRP was applied for 10 min at room temperature followed by 3,3'-diaminobenzidine tetra-hydrochloride (DAB) substrate stain for 10 min at room tem-perature. Gill’s hematoxylin counterstain was applied for 2 min. Slides were then dehydrated and coverslipped.

Images for the figures were captured using a Nikon DS cam-era on a Nikon Eclipse 80i microscope using the 20x objective. Scores for spongiform change were generated after examining defined regions of brain on H&E-stained sections (Table 3). Scores were assigned as follows: +, occasional vacuole indica-tive of aging or spongiform encephalopathy (SE), but diagnosis inconclusive; ++, the presence of crisp, round to oval vacuoles within neurons and/or neuropil, but no even spread of vacuoles, classified as definitive SE; +++, even spread of vacuoles through-out the region with coalescence of some vacuoles, abundant SE. Scores for PrPSc immunohistochemistry were assigned as follows: +, minimal immunoreactivity with multifocal distribution; ++, multifocal to coalescing immunoreactivity affecting up to 25% of the section; +++, immunoreactivity affecting 26–50% of the section; and ++++, severe immunoreactivity affecting greater than 50% of the section examined.

Acknowledgments

This work was supported by grants from the National Institutes of Health (NS041997, AG02132, AG10770 and AI064709 to S.B.P. and PO1 AI 77774-01 to J.A.R.) as well as by a gift from the Schott Foundation for Publication Education. G.T. was sup-ported by a fellowship from the Larry L. Hillblom Foundation. The authors thank Pierre Lessard and the staff of the Hunters Point animal facility for support with the Tg animal experiments; Ana Serban for antibodies; Martha Church, Kevin Hassall, Trudy Tatum for expert technical assistance; the TSE animal caretakers and Hang Nguyen for editorial assistance.

Author Contributions

G.T., J.A.R., M.W.M. and S.B.P. designed the Tg mouse studies; G.T., J.A.R., A.N.H., J.J.G., M.W.M., L.L.W., T.M.S., N.L.J., A.J.Y., A.L. and S.J.D. performed various aspects of the research on sheep, deer or Tg mice; G.T., J.A.R., M.W.M., D.V.G., S.J.D. and S.B.P. analyzed the data; G.T., J.A.R., M.W.M., S.J.D. and S.B.P. wrote the paper. All authors discussed the results and com-mented on the manuscript.

Note

Supplemental material can be found at:www.landesbioscience.com/journals/prion/article/16984

experiment series relative to the ID50

of the SSBP/1 inoculum is then β

1/β

2 - log

10(ID

50 SSBP/1). All calculations were performed

with Stata 10 (Stata Corporation).Protein gel blotting. For protein gel blotting analysis, 10%

(wt/vol) brain homogenates were prepared in PBS by two 45 sec runs at 6.0 m/s with a FastPrep FP120 Cell disrupter (Qbiogene, Inc.). Samples of 5% brain homogenates were incubated with 20 μg/mL of PK (New England Biolabs, Inc.,) for 1 h at 37°C. PK digestion was stopped with 2 mM phenylmethylsulfonyl flu-oride (PMSF) and samples were centrifuged at 100,000x g for 1 h at 4°C. Pellets were resuspended in 10 mM TRIS-HCl (pH 8.0), 0.15 M NaCl, 0.5% (wt/vol) NP-40, 0.5% (wt/vol) sodium deoxycholate. Equal volumes of 2x sodium dodecyl sulfate (SDS) sample buffer were added to the samples before they were boiled for 5 min. For electrophoresis, 30 μl of undigested and PK-digested samples were loaded onto the gels.54 SDS gel electro-phoresis and protein gel blotting were performed using NuPage Novex 4–12% Bis-Tris Midi gels and the iBlot dry blotting sys-tem (Invitrogen). PrP was detected with HuM-P, a PrP-specific humanized Fab derived from a mouse monoclonal antibody,55 that was covalently bound to horseradish peroxidase (HRP) and developed with the enhanced chemiluminescent detection sys-tem (Amersham Biosciences).12

Histopathology. Mouse brains were either instantly frozen after extraction or immersion-fixed in 10% buffered forma-lin and embedded in paraffin. To assess spongiform changes, 8-μm-thick brain sections were stained with hematoxylin and eosin. To evaluate reactive astrocytic gliosis, glial fibrillary acidic protein (GFAP) was immunostained using a rabbit antiserum (Dako). PrPSc from formalin-fixed tissue sections was detected after hydrolytic autoclaving with HuM-P.55

Sheep brain tissues were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 5 μm and stained with H&E for light microscopy and by an automated immunohistologic (IHC) method for detection of PrPSc as described previously in reference 48. Samples from an uninoculated sheep were used as a negative control. Briefly, after deparaffinization and rehydration, tissue sections were autoclaved for 30 min in an antigen-retrieval solution (Target Retrieval Solution, Dako North America, Inc.,) and stained with an indirect, biotin-free staining system contain-ing an alkaline phosphatase-labeled secondary antibody (ultra-view Universal Alkaline Phosphatase Red Detection Kit, Ventana Medical Systems, Inc.,) designed for an automated immunostainer (NexES IHC module, Ventana Medical Systems). The primary antibodies used were F89/160.1.5 and F99/97.6.1, each used at a concentration of 5 mg/ml, and incubation was performed at 37°C for 32 min. For GFAP staining, sections were prepared from par-affin-embedded tissue and adhered to charged slides. After being heated at 60°C for 20 min, deparaffinized and rehydrated, sec-tions were stained using an EnVision G/2 Doublestain System kit


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Salivary prions in sheep and deer