Salivary prions in sheep and deer

doi:10.4161/pri.6.1.16984

Prion. 2012 Jan-Mar; 6(1): 52–61.

Published online 2012 January 1. doi: 10.4161/pri.6.1.16984

PMCID: PMC3338966

Salivary prions in sheep and deer

Gültekin 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 DeArmond,^1,⁷ and Stanley B Prusiner^1,²

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

Corresponding author.

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

Correspondence to: Stanley B. Prusiner; Email: stanley/at/ind.ucsf.edu

Received June 17, 2011; Revised July 28, 2011; Accepted July 29, 2011.

This article has been cited by other articles in PMC.

Abstract

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.

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

Introduction

Scrapie of sheep and chronic wasting disease (CWD) of cervids are highly transmissible, fatal prion diseases that cause neurodegeneration.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 persist 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 experiments 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 transmission 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 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, retropharyngeal 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 asymptomatic, 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 prions 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 expressing ovine prion protein, denoted Tg(OvPrP) mice. Comparison of the resulting incubation times with those from endpoint titrations of scrapie brain homogenates in Tg(OvPrP) mice enabled us 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 similar 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 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.

Table 1

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

Table 2

Neuropathology results for scrapie-infected sheep saliva donors^a

Six of the seven saliva concentrates from sheep with clinical 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. inoculated 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 proteinase K (PK) in protein gel blots (Figs. 1B and S2). Neuropathologic analysis of these brains also showed the presence of spongiform degeneration, accompanied by PrP^Sc deposition and intense astrocytic gliosis (Fig. 2), similar to the neuropathology observed in Tg(OvPrP) mice infected with scrapie prions derived from sheep brain tissue.²⁶

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. infection with scrapie (more ...)

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, (more ...)

To estimate the infectivity of tissues and secretions containing 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 analysis, which estimates the infectious dose (ID₅₀) 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 ID50 U/ml with a 95% confidence interval (CI) between 6.8 and 7.7 log ID₅₀ U/ml. Thus, an entire sheep scrapie brain weighing 109 g contains 10.2 log ID₅₀ 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 ID₅₀ U/ml (Table 1). Considering that an adult ewe weighing 71.2 kg produces 16.6 L of saliva per day,²³^,²⁴ and assuming 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 ID₅₀ 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

Figure 3

Endpoint titration of SSBP/1 prions in Tg(OvPrP) mice. Ten dilutions, ranging from 10⁻¹ to 10⁻¹⁰ as indicated, of a 10% (wt/vol) brain homogenate were i.c. inoculated into 30 groups of 4 mice each. The percentage of mice without neurologic (more ...)

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 biopsies.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. inoculation into Tg(ElkPrP) mice.²⁶ These Tg(ElkPrP^+/+) mice are homozygous for the transgene that expresses ElkPrP with the M132 polymorphism.

Table 3

Transmission of prions in deer saliva to Tg(ElkPrP) mice^a

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 transmitted 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 PrP^Sc upon limited PK digestion (Fig. 4B). Neuropathology showed spongiform degeneration, PrP^Sc deposition and an intense astrocytic gliosis (Fig. 5).

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 (more ...)

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). (more ...)

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 ID₅₀ 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 ID₅₀ U (Eq. S3).

Discussion

We conclude that sheep with clinical scrapie can shed infectious prions in saliva. Because saliva is mostly swallowed, salivary prions may be a continuous source for primary or secondary infection of lymphoid, epithelial, and other susceptible tissues of the GI tract and may thus contribute to peripheral prion replication. Salivary prions that pass through the GI tract may also contribute 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 transmission 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 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 tonsils or the lymph nodes associated with the naso-oropharyngeal cavity.³^,³³ Experiments in prion-infected rodents have shown that the oral and nasal mucosa, including the papillae in the tongue, can harbor prions and may act as potential sources for the horizontal transmission of animal prion diseases since these tissues may release prions during their normal turnover.³⁴^,³⁵ Equally, the tongue, and the oral and nasal mucosa can also act as routes for neuroinvasion.³⁶^–³⁹

Table 4

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

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 ID₅₀ U, which is similar to our value of −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 presence of scrapie prions in buccal swab samples of presymptomatic 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 number 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 infectious prions by small ruminants and cervids, it will be important to determine whether patients with variant Creutzfeldt-Jakob disease (vCJD) who harbor 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 Animal Care and Use Committee, under the CAR “Incubation periods of prion diseases” (AN075039-03). The sheep experiments 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 Disease Center (NADC #3805: “Sheep scrapie-minimum infectious dose”). Mule deer care and research protocols were performed 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 passaged 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 criteria. 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.

Housing, care and inoculation of sheep.

Seven 4-mo-old Cheviot lambs from a scrapie-free flock were obtained for this study and transported to the NADC (Ames, IA) where they were inoculated with scrapie prions. For negative controls, 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 immunohistochemical and protein gel blot analyses.⁴⁷ The infected brains were homogenized by ultrasonication at a final concentration of 20% (wt/vol) in phosphate-buffered 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 procedure described previously in reference 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 developed terminal clinical signs of scrapie as determined by the attending veterinarian. They were examined at necropsy and tissue 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 species.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 collections. 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.

At weaning, mule deer fawns were orally infected with approximately 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 PrP^Sc 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 PrP^Sc accumulation in tonsil and rectal mucosa biopsies,²⁹ indicating successful infection. 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 collected 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 and22). The collected saliva samples were kept frozen in plastic tubes at −80°C until processed. To concentrate the scrapie 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 precipitates 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 6.5 ml because the pellet was resuspended in 300 µl diluent containing 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 ID₅₀ 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 “censored,” 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 i^th comparison, the variable group_k is 1 for animals in the k^th experiment, 0 for animals in the SSBP/1 serial dilution series, and dilution_k gives the dilution in the k^th experiment relative to a 10% homogenate. The nonlinear regression model for the mean onset time in the k^th series, denoted t_k, is log(t_k) = α₀ + α₁ group_k + α₂ dilution_k. The equivalent log titer for the kth experiment series relative to the ID₅₀ of the SSBP/1 inoculum is then α₁ α₂ − log₁₀(ID₅₀ 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 proportional hazards model,52 to represent the hazard as h_k(t) = h₀(t) exp(β₁ group_k + β₂ dilution_k). The equivalent log titer for the k^th experiment series relative to the ID₅₀ of the SSBP/1 inoculum is then β₁/β₂ − log₁₀(ID₅₀ 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 fluoride (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 electrophoresis and protein gel blotting were performed using NuPage Novex 4–12% Bis-Tris Midi gels and the iBlot dry blotting system (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 system (Amersham Biosciences).¹²

Histopathology.

Mouse brains were either instantly frozen after extraction or immersion-fixed in 10% buffered formalin 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). PrP^Sc 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 PrP^Sc 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 containing an alkaline phosphatase-labeled secondary antibody (ultraview 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 paraffin-embedded tissue and adhered to charged slides. After being heated at 60°C for 20 min, deparaffinized and rehydrated, sections were stained using an EnVision G/2 Doublestain System kit (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 tetrahydrochloride (DAB) substrate stain for 10 min at room temperature. 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 camera 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 indicative 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 throughout the region with coalescence of some vacuoles, abundant SE. Scores for PrP^Sc 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 supported 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 commented on the manuscript.

Supplementary Material

Supplementary Material

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