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Transmission and Detection of Prions in Feces

  1. Jiri G. Safar1,2,
  2. Pierre Lessard1,
  3. Gültekin Tamgüney1,2,
  4. Yevgeniy Freyman1,
  5. Camille Deering1,
  6. Frederic Letessier1,
  7. Stephen J. DeArmond1,3 and
  8. Stanley B. Prusiner1,2,4
  1. 1Institute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco
  2. 2Departments of Neurology,University of California, San Francisco, San Francisco
  3. 3Departments of Pathology, University of California, San Francisco, San Francisco
  4. 4Departments of Biochemistry and Biophysics, University of California, San Francisco, San Francisco
  1. Reprints or correspondence: Dr. Stanley B. Prusiner, 513 Parnassus Ave., HSE-774, San Francisco, CA 94143-0518 (stanley{at}ind.ucsf.edu).
 
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Abstract

In chronic wasting disease(CWD)in cervids and in scrapie in sheep, prions appear to be transmitted horizontally. Oral exposure to prion-tainted blood, urine, saliva, and feces has been suggested as the mode of transmission of CWD and scrapie among herbivores susceptible to these prion diseases. To explore the transmission of prions through feces, uninoculated Syrian hamsters (SHas) were cohabitated with or exposed to the bedding of SHas orally infected with Sc237 prions. Incubation times of ∼140 days and a rate of prion infection of 80%–100% among exposed animals suggested transmission by feces, probably via coprophagy. We measured the diseasecausing isoform of the prion protein (PrPSc) in feces by use of the conformation-dependent immunoassay, and we titrated the irradiated feces intracerebrally in transgenic mice that overexpressed SHa prion protein (SHaPrP). Fecal samples collected from infected SHas in the first 7 days after oral challenge harbored ∼60 ng/g PrPSc and prion titers of ∼106.6ID50/g. Excretion of infectious prions continued at lower levels throughout the asymptomatic phase of the incubation period, most likely by the shedding of prions from infected Peyer patches. Our findings suggest that horizontal transmission of disease among herbivores may occur through the consumption of feces or foodstuff tainted with prions from feces of CWD-infected cervids and scrapie-infected sheep.

Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE), scrapie in sheep, and chronic wasting disease (CWD) in cervids are neurodegenerative diseases caused by prions [1]. A wealth of experimental and epidemiological data indicate that variant CJD (vCJD) in humans results from contamination of the food supply (probably mechanically recovered meat) with BSE prions [2–4]. Recent evidence suggests that vCJD also may have been transmitted iatrogenically by means of transfusion performed using prioncontaminated blood [5–7]. Estimation of the dose of prions to which children were exposed during ritualistic cannibalism prompted the hypothesis that another port of entry must exist, perhaps via abrasions of mucous membranes in the oral cavity or eye or via open wounds on the hands [8].

Whereas BSE, vCJD, and sporadic CJD (sCJD) do not seem to be horizontally transmissible by contact, both scrapie andCWDspread naturally from animals in their respective flocks and herds. The rate of transmission of CWD has been reported to be as high as 30%, and it can approach 100% among captive animals in areas of endemicity [9–11]. Despite extensive research, it is unknown how scrapie spreads between sheep or flocks [10]. Vertical transmission of scrapie (between ewes and their offspring) has not been demonstrated [12–14]. Maternal transmission also has been studied in BSE-infected ewes, but transmission of BSE has not been observed either in goats or sheep [15]. Scrapie prions can persist in the environment for up to 3 years after removal of the scrapie-affected flock [16]. Moreover, it is possible to derive a scrapie-free sheep flock from the progeny of a scrapie-afflicted flock, providing that the lambs are kept in a clean environment [17]. Similarly, circumstantial evidence suggests that CWD is transmitted by environmental contamination ofCWDprions [9, 18] or by contact with infected cervids [19, 20].

Oral exposure to prion-tainted blood, urine, saliva, and feces has been suggested as a mode of transmission of CWD and scrapie among herbivores susceptible to these diseases [10, 17, 19, 20]. It is thought that both CWD and scrapie prions are likely to enter the body through gut-associated lymphoid tissues, in Peyer patches in the alimentary tract [10, 21–24]. Moreover, the presence of the infectious isoform of the prion protein (PrPSc) in Peyer patches [10, 21, 25] suggests alimentary shedding of CWD and scrapie prions into feces.

To study the presence of prions in feces, we employed a wellestablished Syrian hamster (SHa) model of oral infection with prions [26]. The study was facilitated by the fact that SHas are cannibalistic [26] and, like many other herbivores, practice coprophagy [27]. When noninfected SHas were cohabitated with SHas orally infected with Sc237 prions, we observed infection rates of 80%–100% within 14 days after oral challenge. Because the rate of transmission of Sc237 prions from bedding was similar to that associated with oral exposure, the data suggest that feces could be the source of infection. We irradiated the feces from orally infected SHas and then measured the concentration of PrPSc by use of conformation-dependent immunoassay (CDI) and the prion titer by use of a bioassay performed in transgenic (Tg) mice overexpressing SHaPrP. We found infectivity titers of ∼106.6 ID50/g and corresponding levels of PrPSc in the first 7 days after oral infection. Excretion of infectious PrPSc continued to occur at lower levels through the asymptomatic period of disease. The data presented in this study provide a mechanism for the horizontal transmission and environmental contamination of prions and may help efforts to control the spread ofCWDand scrapie.

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Materials and Methods

Animals and prion inoculation. Three- to five-week-old female SHas were acquired from Charles River Laboratories and housed in polycarbonate cages with paper bedding (Paper Chip). Transgenes and production of Tg(SHaPrP+/+)7 mice (hereafter referred to as “Tg7 mice”) have been described elsewhere [28]. All studies presented in this article were reviewed and approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco.

For intracerebral inoculation, hamsters and weanling mice were injected with 50 and 30 µL, respectively, of 1% (w/v) brain homogenate prepared from Sc237-infected SHas. Inoculation was performed with a 26-gauge, disposable hypodermic needle inserted into the right parietal lobe.

For intraperitoneal inoculation, 5- to 7-week-old SHas were injected in the right lower quadrant of the abdominal cavity with 100 µL of 1% brain homogenate prepared from Sc237-infected SHas.

For oral challenge, 5- to 7-week-old SHas were placed individually in polycarbonate cages without bedding, assigned to a dietary fast for 18 h, and then offered one-half of the brain from a Sc237-infected SHa in a petri dish. SHas were observed until the brains were consumed entirely.

Inoculated animals were examined daily, and standard diagnostic criteria were used to identify animals affected by prion disease. Animals whose deaths were imminent were euthanized, and their brains were removed for histological and biochemical analysis.

Collection and treatment of feces. Four intracerebrally, intraperitoneally, or orally infected SHas were housed together in standard cages. With the use of sterile forceps, 10 fecal pellets were collected from the bottom of the cage (in the resting area opposite of the corner used for urination) at 1, 8, 22, 43, 64, 85, and 106 days after infection. Different sterile forceps were used for each cage, and only fresh (dark and moist) pellets were selected. Because the intracerebrally inoculated animals developed symptoms starting at ∼70 days after inoculation, their feces were collected only up to 63 days after inoculation. The fecal pellets were stored at −80°C in a plastic container.

To prepare 10% (w/v) homogenate of feces, 1 g of frozen feces was thawed and homogenized in PBS (pH 7.4) by use of a standard Mini Vortexer (VWR Scientific Products). The homogenate was mixed for 10 min and filtered through folded surgical cotton gauze to remove large particles. After final homogenization in a 5-mL syringe equipped with a 21-gauge needle, a 1-mL aliquot was transferred to a sterile glass container and irradiated by a total dose of 2.9 kGy with Cesium-137.

Transmission of prions by cohabitation and bedding. To determine the transmissibility of prions by cohabitation, 17 SHas were orally challenged with Sc237 prions. One ear of each infected animal was surgically notched for identification purposes. One orally infected SHa was placed in a new cage with 3 uninoculated SHas immediately thereafter; 2 h later; or 1, 2, 7, or 14 days after oral challenge (figure 1). All animals were observed for the development of neurological signs of prion disease. To avoid cannibalism, the sick hamsters were removed from the cages as soon as they demonstrated symptoms of prion disease.

Figure 1.
Figure 1.

Experimental setup for the transmission of Sc237 prions by cohabitation. To determine the transmissibility of prions by cohabitation, 17 Syrian hamsters (SHas) were orally challenged with Sc237 prions (top row). One orally infected SHa was placed in a new cage with 3 uninoculated SHas immediately thereafter; at 2 h (0.083 days); or 1, 2, 7, or 14 days after oral challenge. All animals were observed for the development of neurological signs of prion disease.

To evaluate transmission of prions by bedding, 2 SHas infected either orally or intracerebrally with Sc237 prions were housed in standard cages with paper bedding (Paper Chip). After 3.5 or 7 days, both infected hamsters were moved to a new cage, and 4 noninfected hamsters were placed into the cage vacated by the 2 infected animals; the original bedding was maintained (figure 2). This cycle was repeated until the 2 infected hamsters developed prion disease and were euthanized.

Figure 2.
Figure 2.

Experimental setup for the transmission of Sc237 prions by bedding. Two Syrian hamsters (SHas) orally infected with Sc237 prions were housed in standard cages with paper bedding (Paper Chip). After 3.5 or 7 days, both infected hamsters were moved to a new cage, and 4 noninfected hamsters were placed into the cage vacated by the 2 infected SHas; the original bedding of the cage was maintained. This cycle was repeated until the infected hamsters developed prion disease and were euthanized.

Incubation-time assay. The incubation-time assay was performed as described elsewhere [29]. Titers were calculated from the incubation time, by use of either a double-exponential function or power function to describe the association between the incubation time and the prion titer [29, 30].

Sample preparation, CDI, and Western blot analysis. Preparation of brain and fecal samples and detection of PrPSc by CDI and Western immunoblot assay are described in the appendix, which appears only in the electronic edition of the Journal.

Histopathological examination. Brain tissue was either immediately frozen or immersion-fixed in 10% buffered formalin for embedding in paraffin. Sections that were 8 µmthick were stained with hematoxylin-eosin to evaluate vacuolation. Evaluation of reactive astrocytic gliosis was performed by glial fibrillary acidic protein (GFAP) immunostaining with the use of a rabbit antiserum (Dako). Hydrolytic autoclaving pretreatment of the formalin-fixed tissue sections was used to detect PrPSc, as described elsewhere [31].

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Results

Prion Transmission Resulting from Cohabitation with Orally Infected SHas

SHas that were fed Sc237-infected brains were placed in cages with uninfected SHas at 0, 0.1, 1, 2, 7, and 14 days after oral challenge (figure 1). All orally infected hamsters died of prion disease with an incubation time of ∼130 days (table 1). Cohabitation resulted in a transmission rate of 100% when healthy animals were exposed to infected SHas within 2 days after feeding. For uninoculated SHas exposed to infected animals 7 and 14 days after infection, we found transmission rates of 85% and 80%, respectively. From these experiments, we conclude that prions were transmitted from orally infected SHas by direct animal-to-animal contact or by exposure to a high dose of prions in contaminated cages.

Prion Transmission Resulting from Exposure to Bedding of Orally Infected SHas

To test whether exposure to the bedding of infected SHas could result in prion transmission, 2 orally infected SHas were moved out of their cages 3.5 or 7 days after oral challenge, and healthy animals were moved into the cages (figure 2). All orally infected hamsters died of prion disease (mean incubation time, ∼130 days). All SHas that were exposed to the infected bedding, starting at 3.5 and 7 days after feeding, developed prion disease with mean incubation times of 148 and 140 days, respectively (table 2). We conclude that prions were transmitted by exposure to prions in the bedding or cages.

Effect of Irradiation on Sc237 Prions

Because SHas practice coprophagy, we decided to test directly whether the feces of orally infected SHas harbor prions. Because Sc237 prions are exceptionally resistant to gamma radiation, we employed conditions that would selectively inactivate bacteria and viruses, but not prions, in feces: a 2.9-kGy dose (0.29 Mram) has a negligible effect on prion infectivity [32, 33]. The initial cultivation of fecal bacteria after administration of different doses of radiation demonstrated that a dose of 2.9 kGy was sufficient to effectively sterilize the feces. When the 10% fecal extract from uninoculated SHas was irradiated and inoculated intracerebrally into Tg7 mice, it did not induce any noticeable acute or late clinical symptoms; the survival rate for these mice inoculated with feces was comparable to that of mockinoculated animals (figure 3A). Feces that were spiked with 1% brain homogenate infected with Sc237 prions, irradiated, and then inoculated into Tg7 mice were associated with a mean incubation time of ∼55 days, corresponding to a titer of 5.9 ± 0.2 ID50/mL (figure 3A). The same incubation time was obtained when the feces were first irradiated and then spiked with 1% Sc237-infected homogenate (data not shown). In comparison, nonirradiated 1% Sc237-infected brain homogenate inoculated into Tg7 mice was associated with a mean incubation time of ∼44 days, corresponding to a titer of 8.6 ± 0.2 ID50/mL (figure 3A). The difference in the prion titer between infected brain homogenate and feces spiked with infected brain homogenate was 2.7 log ID50/mL, as estimated from the incubation-time assay. We conclude that the decrease in titer was the result of the presence of fecal material.

Figure 3.
Figure 3.

Comparable sensitivity of bioassay and conformation-dependent immunoassay (CDI) for the detection of Sc237 prions diluted in feces of control Syrian hamsters (SHas). A, Bioassay of 1% (w/v) of Sc237-infected brain homogenate (squares), 10% (w/v) irradiated control feces (diamonds), and 10% (w/v) irradiated feces spiked with 1% (w/v) Sc237-infected brain homogenate (circles) in transgenic mice overexpressing SHa prion protein. Data are plotted as the percentage of animals without signs of clinical disease vs. the time after inoculation (expressed in days). The median incubation time is extrapolated from the 50% survival rate in each experiment. The titer was calculated from the incubation time for individual animals and was expressed as the mean ±SE [30]. B, The sensitivity of CDI for the detection of the disease-causing isoform of the prion protein (PrPSc) in control fecal extracts spiked with 1% Sc237-infected brain homogenate and serially diluted in control feces. PrPSc was measured using CDI with recombinant human/mouse chimera of antibody fragment D18 to capture and Eu-labeled monoclonal antibody 3F4 to detect SHaPrP. Total PrPSc (circles) was measured after phosphotungstic acid (PTA) precipitation without protease treatment; rPrPSc (squares) was measured after proteinase K (PK) digestion (50 µg/mL PK for 60 min at 37°C) and PTA precipitation. Each fecal sample was tested 2–4 times, and the results for individual samples were expressed as the mean ±SD. The (D–N) differences (i.e., the differences in the denatured [D] and native [N] aliquots of a sample) in the time-resolved fluorescence (TRF), as measured in counts per minute (cpm), are directly proportional to the PrPSc concentration [34, 35].

CDI Measurement of PrPSc in the Feces of Orally Infected SHas

To calibrate the CDI and to determine the cutoff value, we spiked the feces from uninfected, control SHas with Sc237- infected, SHa brain homogenate. The spiked fecal samples were then diluted in control feces, and they were tested without proteinase K (PK) to measure total PrPSc and with PK to measure protease-resistant PrPSc (rPrPSc), by use of CDI (figure 3B). When considered together with the bioassay data (figure 3A), the limit of detection of PrPSc and rPrPSc in feces corresponds to ⩽100 ID50/mL ⩽30 ID50/mL, respectively. In contrast to PK-treated, Sc237-infected, SHa brain homogenates [34], spiked feces had ∼2-fold higher readings for rPrPSc after PK treatment. This effect may suggest the presence, in feces, of proteins that compete with PrPSc detection or directly interact with PrPSc at the epitope for recombinant antibody fragment D18 (residues 134–158) or 3F4 (residues 109–112).

To measure the excretion of PrPSc from orally infected SHas, we collected the feces of these SHas daily for the first 20 days after infection and then every 3–7 days thereafter (figure 4A), and levels of PrPSc were determined by CDI. A peak in both total PrPSc and the rPrPSc level occurred at 2 days after infection and then gradually decreased over the next 16 days. At 20 days after infection, the PrPSc and rPrPSc levels in the feces of these asymptomatic SHas increased again and fluctuated above the cutoff value until the animals became symptomatic at 116 days after infection (figure 4A).

Figure 4.
Figure 4.

A, Excretion of the disease-causing isoform of the prion protein (PrPSc) and rPrPSc in the feces of Syrian hamsters (SHas) orally infected with Sc237 prions. Each SHa consumed one-half of a brain of an Sc237-infected SHa. Fecal samples were collected daily from day 0 to day 20 and then every 3–7 days until SHas showed symptoms of disease at 116 days. The concentration of prion protein (PrP) isoforms in fecal extracts was measured by conformation-dependent immunoassay (CDI) performed using recombinant antibody fragment HuM D18 to capture and Eu-labeled monoclonal antibody 3F4 to detect SHaPrP. Total PrPSc (circles) was measured after phosphotungstic acid (PTA) precipitation without protease treatment; rPrPSc (squares) was measured after proteinase K (PK) digestion and PTA precipitation, as shown in figure 3. Dashed lines denote the cutoff value, which was calculated as the mean + (3 × SD) for age-matched, control feces. B, Levels of PrPSc in the brains of individual, symptomatic transgenic mice overexpressing SHa prion protein intracerebrally inoculated with irradiated fecal homogenates. At 1, 2, 8, 22, 43, 64, 85, and 106 days after infection, fecal samples were collected from orally infected (po), intraperitoneally inoculated (ip), or intracerebrally inoculated (ic) SHas. As a control (c), age-matched, uninoculated brains were evaluated. The dashed line denotes the cutoff value calculated as the mean + (3 × SD) from age-matched, control brain samples. For both panels, each sample was tested 2– 4 times, and the results for individual samples are expressed as the mean ± SD. The PrPSc concentration was calculated from the (D–N) differences (i.e., the differences in the denatured [D] and native [N] aliquots of a sample) in the time-resolved fluorescence (TRF), as measured in counts per minute (cpm) [34, 35]. The rPrPSc concentration is directly proportional to the (D–N) value and was calculated using a formula published elsewhere [34].

Titration of Feces of Orally Infected SHas in Tg7 Mice

Feces from both intracerebrally and intraperitoneally inoculated SHas transmitted disease to Tg7 mice with low frequency, and the mice became ill late in the incubation period (table 3). In contrast, feces obtained from orally infected SHas at 1 day and 8 days after infection had a titer of 6.6 log ID50/g, which is equivalent to that noted for feces spiked with 1% brain homogenate from SHas infected with Sc237 prions (table 3). The feces collected at later points in time were also infectious: rates of transmission of up to 50% were noted in Tg7 mice. The titers estimated on the basis of the incubation time of infected mice were ⩽2.3 log ID50/g of feces.

Confirmation of Diagnoses

Because one-half of all Tg7 mice homozygous for the SHaPrP transgene array developed disease by ∼460 days of age [36], the clinical diagnoses of Tg7 mice inoculated with feces were verified by CDI, Western immunoblot analysis, standard histopathological examination, and immunohistochemical analysis (figures 4B, 5, and 6). Tg7 mice were classified as testing positive for disease only when they were consistently tested for the presence of Sc237 prions by use of all methods (table 3).

Figure 5.
Figure 5.

Western blot analysis of brain homogenates of transgenic mice overexpressing Syrian hamster (SHa) prion protein intracerebrally inoculated with irradiated fecal homogenates. Inocula were fecal samples collected at 1, 2, 8, 22, 43, 64, 85, and 106 days, as indicated, from intracerebrally inoculated SHas (A) intraperitoneally inoculated SHas (B), and orally infected SHas (C). Phosphotungstic acid (PTA) pellets of proteinase K (PK)-treated brain homogenates (+) were resuspended in 150 µL of 2× SDS sample buffer. Undigested brain homogenates (−) were diluted to 1.2% (w/v) in 1× SDS sample buffer. Equal volumes of undigested and digested samples were boiled for 5 min before electrophoresis. SDS gel electrophoresis and Western blot analysis were performed as described elsewhere [2]. Prion protein (PrP) was detected with the recombinant antibody fragment HuM-P and was developed using the enhanced chemiluminescent detection system (Amersham Biosciences).

Figure 6.
Figure 6.

Neuropathological and immunohistochemical analyses of brain sections obtained from transgenic mice overexpressing Syrian hamster (SHa) prion protein intracerebrally inoculated with irradiated 10% fecal filtrate. Inocula were fecal samples obtained from Syrian hamsters (SHas) at 1 day (G–I) and 106 days (J–L) after oral feeding of Sc237 prions, as well as from SHas at 42 days after intracerebral inoculation with Sc237 prions (M–O). As controls, Tg7 mice were inoculated with PBS (A–C) or irradiated feces from uninfected SHas (D–F). The hippocampal CA3 region (A–C), hippocampal CA1 region (G–I), ventral pontine nucleus (J–L), and thalamus (M–O) are shown. The bar in panel J denotes 25 µm and applies to all of the prion protein–stained sections by the hydrated autoclaving method (left column). The bar in panel L denotes 50 µm and applies to all of the hematoxylin-eosin– and glial fibrillary acidic protein–stained sections (middle and right columns, respectively).

Table 1.
Table 1.

Cohabitation of uninoculated Syrian hamsters (SHas) with orally infected SHas, starting at different time points after feeding.

Table 2.
Table 2.

Exposure of uninoculated Syrian hamsters (SHas) to the bedding of orally infected SHas, starting at different time points after feeding.

Table 3.
Table 3.

Findings from a bioassay of feces collected at different time points from Syrian hamsters infected with Sc237 prions.

CDI and Western immunoblot analysis. Clinical diagnoses were confirmed in 31 (79%) of 39 samples, by use of both the CDI (figure 4B) and Western blot analysis (figure 5). Despite being associated with similar incubation times in Tg7 mice, feces collected from intraperitoneally inoculated SHas resulted in PrPSc levels in the brains of infected mice that were ∼10-fold lower than the levels resulting from feces collected from intracerebrally inoculated SHas. CDI data for the brains of Tg7 mice inoculated with feces collected from orally challenged SHas also could be separated into 2 groups. In one group, a high concentration of rPrPSc was found in Tg7 mice with short incubation times; in the other group, brain samples obtained from Tg7 mice that had longer incubation times harbored ∼10-fold less rPrPSc. Whether these differences are due to (a) age-accelerated disease associated with lower levels of PrPSc or (b) the development of new prion strain characteristics caused by replication in the gastrointestinal lymphoreticular system remains to be established.

Neuropathological examination. In asymptomatic, 544- day-old control Tg7 mice intracerebrally inoculated with PBS, we found oval and round intracellular deposits of normal, cellular PrP (PrPC) that varied from 2 to 8 µm (figure 6A). More than 95% of the deposits were located in the gray matter neuropil away from nerve cell bodies; some deposits were located adjacent to or within nerve cell bodies, and sparse deposits were found in the white matter. PrP deposits did not bind Thioflavin S and, therefore, were not a form of amyloid. Neither vacuolation nor nerve cell loss was detected by hematoxylin-eosin staining (figure 6B), and no reactive astrocytic gliosis was identified by GFAP immunohistochemical analysis (figure 6C). In age-matched mice intracerebrally inoculated with irradiated feces from an uninfected hamster, we did not observe any residual material or changes that could be associated with the injection of fecal material (figure 6D and 6F). We conclude that the PrP deposits in aged Tg7 mice did not have the tinctorial characteristics of PrPSc and were induced by overexpression of PrPC.

In contrast, we found widespread, moderate-to-severe neuropathological abnormalities in a symptomatic Tg7 mouse intracerebrally inoculated with irradiated fecal filtrate collected from SHas 1 day after oral challenge. In all gray matter regions, irregularly sized deposits of PrPSc were easily distinguished from the background of PrPC deposits on the basis of their slightly blurred edges (figure 6G). We found Thioflavin S–positive amyloid plaques located between the hippocampus and the corpus callosum. Small numbers of gray matter vacuoles (figure 6H) and hyperintense reactive astrocytic gliosis (figure 6I) were observed. Similar neuropathological changes were seen in Tg7 mice intracerebrally inoculated with fecal material collected from SHas 105 days after oral challenge; PrPSc deposits were primarily found in the base of the pons (figure 6J), in association with vacuolar degeneration of gray matter (figure 6K) and severe reactive astrocytic gliosis (figure 6L).

Fecal material that was collected from SHas 42 days after intracerebral inoculation and that was intracerebrally injected into Tg7 mice resulted in disease after 362 days. Brain sections showed moderately severe neuropathological changes in most regions, and mild changes were noted in the neocortex and hypothalamus. Although PrPSc deposits were punctate and relatively difficult to distinguish from PrPC aggregates, the presence of larger, plaquelike masses verified that the depositions were PrPSc (figure 6M). Showing moderate fluorescence with Thioflavin S, these masses likely represent primitive PrP amyloid plaques (data not shown). Gray matter vacuoles were detected only in the thalamus. Moderate reactive astrocytic gliosis was detected in most regions, with the exceptions noted above (figure 6O).

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Discussion

Studies of the mechanism of natural transmission and spread of prions are important to understanding how prion reservoirs form in nature. In most instances, prions are incapable of infecting another host by contact or cohabitation; sheep farmers and veterinarians in contact with scrapie or family members in daily contact with patients with CJD appear to be immune to prion infection [37]. In contrast, there are high incidences of CWD in cervids and scrapie in sheep in areas of endemicity [10, 19]. The source of environmental contamination and whether these natural reservoirs are maintained by a high rate of transmission from animal to animal or by contact with the contaminated environment are subjects of debate [9, 10, 19].

The data presented in the current study clearly demonstrate that laboratory prions are transmissible by cohabitation with orally infected SHas and that the source of infection is prion-infected feces. High PrPSc levels and prion titers were detected within 7 days after feeding prion-infected brain to SHas, indicating that prions pass intact through the digestive tract. The lower transmission rates and long incubation times for disease in Tg7 mice inoculated with feces obtained later (at >10 days after infection) suggest that the gastrointestinal lymphatic system (tonsils, Peyer patches, or mesenteric lymph nodes) of the SHas was infected [38]. Indeed, immunostaining for the detection of PrPSc in mesenteric lymph nodes, mucosal lymphoid follicles, and Peyer patches was observed from the earliest time point available at 69 days after inoculation of SHas orally infected with Sc237 prions [38]. When this information is considered along with the results of the present study of SHas and with observations regarding both naturally occurring CWD and scrapie [10, 21, 25], alimentary shedding of prion-infected cells or cell fragments into feces seems to be a likely means of transmission.

The observation that Tg7 mice intracerebrally inoculated with irradiated, prion-free feces showed no acute toxic symptoms was surprising (figure 6F). In contrast, Tg7 mice that were intracerebrally inoculated with irradiated fecal filtrate obtained from SHas infected with Sc237 prions demonstrated all the classic characteristics of prion infection, but the distribution and intensities of neuropathological changes varied greatly. Unexpectedly, we found neuropathological changes in the pontine base and cerebellum and few or no changes in other regions of the brain (figure 6J–L). Whether this change in anatomical targeting is a result of the presence of feces in the inoculum or whether it reflects a permanent shift in the Sc237 strain characteristics caused by passage through the intestinal lymphoreticular system remains to be established.

The horizontal spread of CWD and scrapie, as well as their transmission by contaminated environments, implicates saliva, feces, and/or urine as vectors of infectivity. In a recent study, saliva from CWD-positive deer transmitted prions to other deer within 12 months of oral challenge. When 2 white-tailed deer were orally challenged with feces and urine obtained from CWD-infected deer, no instances of transmission were observed [20]. The 2 deer expressed the G/S polymorphism at PrP codon 96, which has been shown to confer longer incubation times (>18 months) even for high-titer CWD inocula prepared from brain homogenate [39]. Given the small number of animals, expected low titers, and 12 months of observation in that study [20], conclusions about CWD infectivity in feces must be tempered.

Although our studies provide evidence of prion transmission through feces, reports of prion infectivity in urine have been contradictory. Two studies reported the detection of PrP and low levels of prions in urine [40, 41]. However, we and other investigators have demonstrated that the reported protease-resistant PrP band detected in Western blots of the urine of diseased hamsters results from the cross-reactivity of the anti–mouse IgG antibody with IgG light chains and, possibly, heavy chain fragments; therefore, this band is not likely to be PrP [42–44]. Moreover, urine was collected in metabolic cages, and no attempt was made to separate feces until the clarification spin [41]. Perhaps, as our studies demonstrate, the considerable levels of prions in feces inadvertently contaminated the urine.

Considering the long-term survival of prions in soil [45, 46], the data presented in the current study have raised imperative questions about the safe disposal of excrement from BSEinfected cattle and animals with CWD and scrapie, especially during the early, asymptomatic stage of disease. The methods of disposal of excrement from patients with vCJD and sCJD in a hospital or home setting also should be reexamined. If the presence of prions in feces is verified by bioassays or CDI, safe disposal protocols will have to be implemented. In natural reservoirs of scrapie and CWD, treatment of the feces before disposal should help to minimize long-term environmental contamination with prions.

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Acknowledgment

We thank the staff of the Hunters Point Animal Facility for their expert Syrian hamster and mouse studies.

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Appendix

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Supplementary Material and Methods

Sample preparation. Brain tissue slices weighing 250–350 mg were homogenized to a final 15% (w/v) in 4% (w/v) sarkosyl in PBS (pH 7.4) by undergoing three 75-s cycles in a reciprocal homogenizer Mini-BeadBeater-8 (BioSpec Products). Feces weighing 250–350 mg were homogenized to a final 15% (w/v) in 4% (w/v) sarkosyl in PBS (pH 7.4) containing 0.05% NaN3 by undergoing four 75-s cycles in a reciprocal homogenizer. Homogenates were diluted to a final 5% (w/v) by use of PBS containing 4% (w/v) sarkosyl, 1% (w/v) Prnp0/0 mouse brain homogenate (for fecal samples only), and 50 µg/mL proteinase K (unless stated otherwise), and they were incubated on the shaker for 60 min at 37°C. After a clarification spin in a drum rotor (Jouan) at 500 g for 5 min at room temperature (RT), the samples were mixed with stock solution containing 4% sodium phosphotungstate (NaPTA) and 170 mmol/L MgCl2 (pH 7.4), to obtain a final concentration of 0.32% NaPTA. After undergoing incubation on a rocking platform for 60 min at 37°C, the samples were centrifuged in a Jouan MR23i centrifuge at 14,000 g for 30 min at RT. The resulting pellets were resuspended in 83 µL of H2O containing protease inhibitors (0.5 mmol/L phenylmethylsulfonyl fluoride; 2 µg/mL each aprotinin and leupeptin), and they were assayed by conformation-dependent immunoassay (CDI) or Western immunoblot analysis.

Sandwich CDI for the detection of SHaPrPSc (the disease-causing isoform of the prion protein [PrP] in Syrian hamsters [SHas]). The principle, development, calibration, and calculation of the PrPSc concentration from CDI data have been described in detail elsewhere [34, 35, 47]. The concentrations of PrP isoforms in fecal extracts were measured using CDI with recombinant human/mouse chimera of antibody fragment (recFab) D18 [48] to capture and Eu-labeled monoclonal antibody 3F4 to detect SHaPrP after phosphotungstic acid (PTA) precipitation with or without protease treatment. As a standard, we used denatured recombinant SHaPrP (residues 90–231). Each plate contained positive and negative controls prepared from pooled Sc237-infected or uninfected SHa brains. The results were expressed as either the ratio or difference in the time-resolved fluorescence (TRF; measured as counts per minute) of denatured (D) and native (N) aliquots of a sample. For simplicity, the values are referred to as the D/N ratio or the (D–N) difference. The concentration of protease-resistant PrPSc is directly proportional to the (D–N) value and was calculated using a formula published elsewhere [34].

Western blot analysis. The PTA pellets precipitated from brain homogenates were mixed with an equal volume of SDSloading buffer and boiled for 5 min; 30-µL samples were loaded on 12.5% Tris-glycine SDS-PAGE Novex gels (Invitrogen). Each of 2 duplicate Western blots was incubated with 1µg/mL recFab HuM-P for 2 h at RT [35]. After extensive washing, both blots were developed with peroxidase-labeled, anti–human Fab secondary antibody and by the enhanced chemiluminescence system, as described elsewhere [49].

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Footnotes

  • Potential conflicts of interest: none reported.

  • Financial support: National Institutes of Health (grants AG02132, AG010770, NS22786, and NS14069); G. Harold and Leila Y. Mathers Foundation; Sherman Fairchild Foundation.

  • Received October 9, 2007.
  • Accepted November 15, 2007.
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