This study was done in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Washington, DC, USA) and the Guide for the Care and Use of Laboratory Animals in Research and Teaching (Federation of Animal Science Societies, Champaign, IL, USA). The protocol was approved by the Institutional Animal Care and Use Committee at the National Animal Disease Center (protocol number: ARS-2017-628).
Inoculum sources and preparation
The inocula for this experiment were derived from experimental studies and field isolates. The agent of transmissible mink encephalopathy was previously passaged in cattle (bTME) three times  and passaged to sheep . Two separate groups of ovine passaged bTME (o-bTME) were derived from sheep with different prion protein genotypes: VRQ/VRQ (o-bTMEVV) and VRQ/ARQ (o-bTMEAV). Classical bovine spongiform (C-BSE) encephalopathy and L-type BSE (L-BSE) samples were obtained from field cases in the U.S. (2003) and France (2005) , respectively. The final inocula were prepared as 1% (w/v) homogenates using sterile phosphate-buffered saline.
Transgenic mice, inoculation, endpoints, and sample processing
We used a transgenic mouse model to compare the pathologic phenotypes of bTME, o-bTME, C-BSE, and L-BSE. Transgenic mice overexpressing bovine PrPC (TgBovXV) were obtained from the Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health, Greifswald—Isle of Riems, Germany) . Experimental groups included 18-21 mice per inoculum group; a minimum sample size of 15 mice is recommended for studies with expected long incubation periods . Mice were anesthetized with isoflurane and inoculated intracranially with 20 mL of a 1% w/v brain homogenate derived from clinically diseased animals previously confirmed to have a TSE by EIA, western blot, and anti-PrPSc IHC. Approval from the Institutional Animal Care and Use Committee was procured prior to conducting this experiment (protocol number ARS-2017-628).
Following inoculation, all mice were housed in a biosafety level 2 or 3 facility (3 for BSE inoculated animals) and fed a pelleted rodent ration with access to water ad libitum. They were co-housed in cages specific to their inoculum group. Mice were examined daily for potential signs of prion disease including poor hygiene/haircoat, ataxia, circling, or inability to right position. Upon discovery of clinical signs, the animals were humanely euthanized, and routine samples were collected. When death resulted from intercurrent disease, samples were also collected, but specific criteria (see survival analysis) were used to determine which mice were used to calculate incubation periods. At the predetermined experimental endpoint of approximately 700 days, any unaffected/asymptomatic mice were humanely euthanized. The methods of euthanasia approved and used for these experiments were inhalation of carbon dioxide gas or anesthesia with isoflurane followed by decapitation in accordance with the AVMA Guidelines for the Euthanasia of Animals and the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Washington, DC, USA). An enzyme immunoassay (EIA) was used as a screening test for prion disease for all mice. To complement the results of diagnostic EIA, western blotting, and hematoxylin and eosin stained sections of brain confirmed the presence of spongiform encephalopathy and neuropathology consistent with a diagnosis of TSE.
For sample collection, a 2/3 longitudinal section of the brain was fixed in 10% buffered neutral formalin, and the other 1/3 was frozen for downstream EIA and guanidine hydrochloride fibril stability analyses. Only brains from recently euthanized mice were used for microscopic examination. Formalin fixed brains were transected at the levels of the frontal cortex, hippocampus, midbrain, and medulla oblongata resulting in five sections  that were paraffin embedded and sectioned to 4 µm thickness. Sections were placed on glass slides and stained with hematoxylin and eosin.
Enzyme immunoassay (EIA) was carried out similarly to previously described methods [13, 60] using a commercially available enzyme-linked immunoassay (HerdChek®, IDEXX Laboratories Inc., Westbrook, ME). Frozen brain samples were prepared as a 20% (w/v) tissue homogenates and treated with proteinase K. From that point, the assay was completed according to kit instructions. Cut-off numbers were determined with a negative control as per the kit instructions; values greater than the mean optical density (O.D.) of negative controls + 0.180 were considered positive.
To calculate the mean incubation period (IP) in mice that died prior to the experimental endpoint, we averaged the survival times of EIA positive mice and removed outliers beyond three standard deviations from the mean. Any mice that died preceding three standard deviations from the mean were not included in the IP calculation since their incubation time was artificially shortened due to intercurrent disease. However, the attack rates (AR) were calculated by including all EIA positive mice in the numerator. The denominator of AR was determined after censoring EIA negative mice that died earlier than three standard deviations less than the mean IP (early intercurrent disease). Analyses for all experiments were performed using Microsoft Excel (Microsoft Office, Redmond, WA) and GraphPad Prism 7 (GraphPad Software, San Diego, CA).
Transmission efficiency calculations
In order to compare the transmission efficiencies (TE) of different TSE isolates to transgenic mice, we utilized a computational model that incorporates attack rate (AR) and incubation period (IP) . Determination of the IP multiplier was performed in alignment with the method described by Nonno and colleagues  based on the duration of survival in TSE positive mice (incubation period). There were six possible IP multiplier categories with corresponding values of 1, 0.83, 0.67, 0.50, 0.33, and 0.17 that were assigned based on the average incubation period in days: <200, 200-299, 300-399, 400-499, 500-599, and >600, respectively.
Microscopic examination and lesion profiling
Spongiform change was evaluated with hematoxylin and eosin stained sections of brain using brightfield microscopy. The severity of vacuolation was scored on a scale from 0-5 in predefined grey matter locations as previously described . The score (magnitude of vacuolar change) was plotted against neuroanatomic location to construct a lesion profile. Grey matter areas included the medulla (G1), cerebellum (G2), midbrain (G3), hypothalamus (G4), thalamus (G5), hippocampus (G6), para terminal body (G7), and cerebral cortex (G8 and G9). White matter in the cerebellar peduncle (W1), lateral tegmentum (W2), and the internal capsule (W3) were also evaluated for spongiform change [61, 62] on a scale from 0-3. A minimum of six mice were scored per group based on a single observer. Mean scores were plotted with error bars representing the standard error of the mean (GraphPad Prism 7, GraphPad Software, San Diego, CA). Differences between the means greater than one were generally considered to indicate substantial differences in vacuolation.
Immunohistochemistry for PrPSc
In order to assess the patterns of PrPSc accumulation in the brains of mice inoculated with different TSE agents and strains, we performed immunohistochemistry on formalin fixed paraffin embedded brain sections. Slides were rehydrated with xylene and ethanol. Antigen retrieval was performed in TE buffer (10 mM Tris Base, 1 mM EDTA, 0.05% Tween 20, pH 9.0) held at 121° C for 20 minutes in an autoclave. Slides were then treated with 10% formic acid for 10 minutes. The rest of the protocol was performed on a BOND-Max automated immunohistochemistry stainer (Leica Biosystems, Buffalo Grove, IL) with 3-6 washes between steps. Proteinase K was applied for 7 minutes (20 mg/mL in TE buffer, pH 8.0). Non-specific protein binding was blocked by incubating with Background Buster for 30 minutes (Innovex Biosciences, Richmond, CA). The primary antibody, 6C2 (WBVR, Lelystad, Netherlands), was diluted 1:2000 in a commercial antibody diluent (Agilent-Dako, Santa Clara, CA) and applied to the slides for 15 minutes. Slides were developed with a BOND Polymer Refine Detection kit (Leica Biosystems, Buffalo Grove, IL) and counterstained with Mayer’s modified hematoxylin (Abcam, Cambridge, MA) diluted with water (1:4). Tissues were counterstained with hematoxylin.
Fibril stability determination
Determination of the PrPSc fibril stability was conducted using a commercially available enzyme immunoassay/EIA kit (HerdChek®, IDEXX Laboratories Inc., Westbrook, ME ) as previously described . Three mice were selected for analysis from each treatment group based on the proximity of their survival time to the group mean incubation period. Whole brain homogenates were normalized to an EIA absorbance in the range of 0.8 to 1.5 and incubated in guanidine hydrochloride (GdnHCl) at the indicated concentration for 1 hour. The samples were then diluted to 0.25 M GdnHCl, and the level of PrPSc remaining at each concentration was determined using EIA. Each mouse sample was analyzed in triplicate to ensure repeatable measures. The fraction of remaining fibril was determined by normalizing the O.D. values to the 0.25 M GdnHCl treatment point. Fibril stability is reported as the average concentration of GdnHCl at which 50% of the PrPSc remains in the fibril form ([GdnHCl1/2]). Tukey’s multiple comparison test for ordinary one-way ANOVA was used to determine the significance (alpha=0.05) of differences in the mean fibril unfolding [GdnHCl1/2] for each group (GraphPad Prism 7, GraphPad Software, San Diego, CA).
In order to characterize the molecular properties of each inoculum group, we performed western blots to separate the three glycosylation states of PrPSc. Samples had previously been homogenized to 20% (w/v) in PBS. For digestion of proteinase K-sensitive PrP, 0-5 mL of PBS and 1.5 mL of PK (1 mg/mL, ThermoFisher Scientific, Waltham, MA) were added to 15-20 mL of sample homogenate to create a 21.5 mL reaction. Samples were incubated at 37° Celsius for 1 hour with constant mixing at 800 rpm. After incubation, the total volume of the solution was increased to 100 mL PBS and PK digestion was stopped with 1.5 mL of Pefabloc® (100 mg/mL) (Sigma-Aldrich, St. Louis, MO). Acetone precipitation of proteins was performed to enhance western blot banding patterns. Acetone was chilled to -20° Celsius. Then 400 mL was mixed with 100 mL of each digested sample and incubated at -20° Celsius for 1.5 hours. The precipitated samples were spun at 15,000 rcf for 10 minutes. The supernatant was decanted, and the pellet was allowed to dry for 5-10 minutes. The pellets were resuspended in 30 mL of 1x loading buffer with 1.5 mL of b-mercaptoethanol. Samples were boiled at 100° Celsius for 5 minutes. NuPage 12% Bis-Tris precast gels (ThermoFisher Scientific, Waltham, MA) were loaded with 0.25 – 4 mg tissue equivalents of brain material per well. Gels were run at 200 volts for 45 minutes in 1x MOPS running buffer. Proteins were transferred to low fluorescence PVDF membrane in a 10% methanol transfer buffer for 45 minutes at a constant 25 volts. The membrane was probed with primary anti-PrPSc antibody 6H4 (diluted to a final concentration of 0.09 µg/ml, ThermoFisher Scientific, Waltham, MA), and incubated overnight at 4° Celsius. The remaining steps in the procedure were similar to previously described methods . For the secondary incubation, we used a biotinylated anti-mouse antibody for 1 hour (diluted to a final concentration of 0.1 µg/ml; Biotinylated anti-mouse IgG, Amersham Biosciences, USA) followed by incubation with streptavidin conjugated to horseradish-peroxidase for 1 hour (diluted to a final concentration of 0.1 µg/ml; Streptavidin horseradish-peroxidase conjugate, Amersham Biosciences, USA). Horseradish peroxidase substrate (Pierce ECL-Plus, ThermoFisher Scientific, Waltham, MA) was used to develop a detectable signal that was imaged with a G:BOX gel imaging system (G:BOX Chemi-XT4, Syngene, Frederick, MD). Western blot analyses were performed with Image LabTM Version 6.0.1 for Mac (Bio-Rad Laboratories, Inc, Hercules, CA).