The laboratory and animal experiments were conducted in Biosafety Level 2 spaces that were inspected and approved for importing prion agents by the US Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services. The studies were 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 Agricultural Animals in Research and Teaching (Federation of Animal Science Societies, Champaign, IL, USA). The protocols were approved by the Institutional Animal Care and Use Committee at the National Animal Disease Center (protocol numbers: 3636 and 3985), which included a biosafety review. Cattle were monitored twice daily for the appearance of clinical signs suggestive of prion disease such as aggression, behavior changes, decreased feed intake, loss of body condition, ataxia, prolonged recumbency, or inability to rise. All inoculated animals developed the clinical signs specifically described in Table 1 and no animals died prior to the experimental endpoint. To ensure a humane endpoint, animals were euthanized if they demonstrated clinical signs of prolonged anorexia (>24 h) or recumbency (>12 h). Animals exhibiting behavioral or postural changes consistent with TSE were examined by a veterinarian. Upon veterinary confirmation of unequivocal clinical signs of TSE, affected cattle were killed with an intravenous injection of pentobarbital sodium according to the manufacturer’s directions and necropsied. Any other concurrent diseases were treated under the direction of a veterinarian.

Cattle (n = 12) were intracranially inoculated with 1 ml of a 10% (wt./vol.) brain homogenate from a cow diagnosed with classical BSE in the US in 2003 as previously described (20). The PRNP gene of all inoculated animals was sequenced. The predicted amino acid sequences from inoculated cattle were similar to each other and consistent with previously reported cattle sequences (21). Brain samples from various regions including brainstem at the level of the obex, cerebellum, midbrain, thalamus, and cerebrum were obtained from cattle in the following experimental groups: (1) wild-type cattle intracranially sham-inoculated with normal brain homogenate (negative controls), (2) wild-type cattle intracranially inoculated with classical BSE (22), or (3) knockout cattle (PRNP^−/−) intracranially inoculated with the transmissible mink encephalopathy (TME) agent. Homogenates were prepared from brainstem sections of TSE-free cattle or cattle with clinical signs of TME (23–25) or classical BSE by homogenization with 1.0 mm silica beads in 1× Dulbecco’s PBS pH 7.4 to produce a 10% w/v homogenate. Samples were centrifuged briefly at 1,000 g for 2 min and supernatants were separated and stored at −80°C in aliquots. The sham-inoculated negative control animal was housed separately from the other cattle in this study. All animal donor information including TSE isolate inoculated, source of inoculum, incubation periods, and description of clinical signs are shown in Table 1.

After fixation in 10% neutral buffered formalin, tissues were embedded in paraffin and processed by routine methods for microscopic examination after staining by hematoxylin and eosin or by immunohistochemical methods as described previously (26). Sections of cerebrum, thalamus, midbrain, and brainstem at the level of the obex were assessed for spongiform change and PrP^Sc accumulation. After deparaffinization and rehydration, sections (4 µm on charged slides) were autoclaved for 20 min in antigen retrieval solution (DAKO Target Retrieval Solution, DAKO Crop., Carpinteria, CA, USA), and stained with an indirect, alkaline phosphatase-labeled secondary antibody (ultraview Universal Alkaline Phosphatase Red Detection Kit, Ventana Medical Systems, Inc., Tucson, AZ, USA). The primary antibody was mAb F99/97.6.1 (27) used at 10 µg/ml for a 32 min incubation at 37°C. Slides were counterstained with Gill’s hematoxylin and bluing agent (Ventana Medical Systems, Inc., Tucson, AZ, USA) prior to coverslipping.

All brain tissues including one sham-inoculated negative control, two TME-infected PRNP knock out cattle, three BSE-infected cattle: two with clinical signs (animal No. 2 and No. 3) and one without clinical signs (animal No. 1) were run on the Western blot (WB). Each sample was digested with 50 µg of proteinase K (PK) for 1 h at 42°C and separated by SDS-PAGE on 12% polyacrylamide minigels (Invitrogen) and then transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) for 45 min at 30 V. Bovine serum albumin (3%) in Tris-buffered saline was used to block the membranes for 1 h and then membrane was incubated with mouse anti-PrP monoclonal antibody 6H4 as the primary antibody at 1:10,000 dilution (0.1 µg/ml) for another hour. Then, a biotinylated sheep antimouse secondary antibody at 0.05 µg/ml and a streptavidin–horseradish peroxidase conjugate were used in conjunction with a chemiluminescent detection system (Pierce ECL plus, Thermo Fisher) and visualized on an imaging system capable of detecting luminescence.

Enzyme immunoassay was performed to determine the relative amount of misfolded protein in brain homogenates from all animals including one sham-inoculated, two TME-infected PRNP knock out cattle, three BSE-infected cattle: two with clinical signs (animal No. 2 and No. 3) and one without clinical signs (animal No. 1) using an IDEXX HerdChek BSE EIA kit without treatment of PK. The cutoff value was determined by the negative control sample provided by the manufacturer and the optical density value was around 0.08 ± 0.005. Samples were considered positive if the optical density value was over 0.15.

Brain homogenates (10% w/v) prepared in PBS were digested or treated with different concentrations of PK (0–0.5 mg/ml; VWR, Visalia, CA, USA) for 1 h at 37°C. 1× pefabloc proteinase inhibitor (VWR, Visalia, CA, USA) was added to terminate the reaction. The relative amount of misfolded protein was measured by IDEXX HerdChek BSE Kit (see above). Each sample was performed in triplicate.

Escherichia coli [BL21(λDE3)] transformed with the pET28a vector harboring the wild-type bovine PrP gene (amino acids 25–241; GenBank accession number: DQ875147.1) was grown, and the bovine prion protein was expressed and purified as described by Vrentas et al. (28). The concentration of purified pooled protein was measured by UV spectroscopy and calculated from the absorbance at 280 nm using an extinction coefficient of 63,495 M/cm as calculated for wild-type protein.

Real-time quaking-induced conversion reactions were performed as previously described (29). The RT-QuIC reaction buffer was composed of 10 mM phosphate buffer (pH 7.4), 300 mM NaCl, 0.1 mg/ml recombinant bovine prion protein, 10 µM ThT, 1 mM ethylenediaminetetraacetic acid tetrasodium salt, and 0.001% SDS. A volume of 98 µl of the RT-QuIC reaction buffer was loaded into each well of a black 96-well plate with a clear bottom (Nunc, Thermo Fisher Scientific) and 2 µL of brain homogenate dilution was added to test for seeding activity. Brain homogenate (10% w/v) was prepared as previously described (11) and stored at −80°C.

The plate was sealed and incubated in a BMG FLUOstar Omega plate reader at 42°C for 100 h with cycles of 15 min shaking (700 rpm double orbital pattern) and 15 min rest. ThT fluorescence measurements (460 nm excitation and 480 nm emission) were taken every 15 min, with the gain set at 1,400. All reactions for each sample were performed in eight replicates (quadruplicates in two independent RT-QuIC assays). ThT fluorescence data are displayed as the average ThT fluorescence of four technical replicates for each time point and, to be considered positive, the ThT fluorescence of at least two replicate reactions must be positive. The predefined positive threshold was calculated as 10 SDs above the mean fluorescence of normal cattle brain homogenates. Previously described criteria were applied for classification of positive samples of RT-QuIC (18, 30, 31).

To further characterize the potential infectivity of brain material from animal No. 1 and allow for comparison with RT-QuIC results, bioassays were conducted in TgBovXV (32) mice under isoflurane anesthesia as previously described (33). Inocula were derived from the brainstem of subclinical bovine No. 1 (10% w/v in PBS) or the original classical BSE inoculum (1% w/v in PBS) (34). Each mouse was inoculated with 20 µl intracranially. Mice were monitored daily for the development of clinical signs by animal care staff. When signs suggestive of prion disease such as ataxia, listing or rolling gate, pelvic limb paresis, lethargy, or poor grooming with urine stained fur were recognizable by observation, animals were humanely euthanized and their brains prepared for analysis by EIA as described above. The attack rate was defined as the percentage of mice from an experimental group that had a positive EIA result. Incubation periods are expressed as the mean days postinoculation (days PI) for mice with positive EIA results. All animals that died due to intercurrent disease or without the development of clinical signs 2 SDs or less prior to the average incubation time were included in the calculation of attack rate. Using these criteria to calculate attack rate, 18 mice were used for the classical BSE group and 16 mice were included in the No. 1 inoculation group.

The incubation period for classical BSE in cattle is reported here as the time from inoculation to the time when unequivocal signs of clinical disease were present. Clinical signs of disease included abnormalities in gait or stance, moderate to severe ataxia, and hyperreaction to stimuli such as noise or movement. The average incubation period was approximately 22 months (22). One animal (No. 1) was euthanized at the termination of the study at 31 months without developing clinical signs (Table 1) despite receiving the same inoculum by intracranial inoculation without any noted deviations in procedure. Tissues from this animal were subjected to additional studies in an effort to explain the failure to develop clinical signs.

In the cattle clinically affected with BSE, PrP^Sc immunoreactivity was detected in all brain regions examined and corresponded with spongiform change. Spongiform change was most severe in the brainstem (nucleus of the solitary tract and nucleus of the spinal tract of the trigeminal nerve (Figure 1A1), caudal midbrain, and hypothalamus. PrP^Sc immunolabeling in the steer No. 2 (clinically affected with BSE) included granular, linear, aggregated, intraneuronal, and stellate labeling types. There was moderately strong immunolabeling of the cerebrum (neocortex) with prominent stellate and intraglial labeling and less granular and intraneuronal labeling compared to the thalamus, midbrain) and brainstem (Figure 1A2). There was no evidence of spongiform change or PrP^Sc immunoreactivity in any of the other groups represented: inoculated with the BSE agent but prior to the onset of clinical signs (steer No. 1, Figures 1B1,B2); knockout cattle (PRNP^−/−) inoculated with the TME agent, or a sham-inoculated control animal (Figures 1C1,C2).

Samples of brainstem at the level of the obex were collected from representative cattle. WBs were done using the monoclonal antibody 6H4 to demonstrate PrP^Sc. WB results are shown in Figure 2. WB confirmed evidence of PrP^Sc in cattle with clinical signs (Nos. 2 and 3), but failed to demonstrate PrP^Sc in cattle necropsied prior to the onset of clinical signs (No. 1), knockout cattle inoculated with ME (Nos. 4 and 5), or a sham-inoculated steer (No. 6).

Enzyme immunoassay was initially performed to on brainstem samples from all BSE-inoculated cattle to determine the relative amounts of misfolded prion protein present. Similar to IHC and WB, no misfolded prion protein was detected in the brainstem of steer No. 1, whereas high levels were detected in the brainstem of cattle with clinical signs (steers No. 2 and No. 3; see Table 2).

Steer No. 1 was inoculated with brain homogenate of known infectivity, but brainstem samples tested negative by EIA. Therefore, EIA was performed on additional brain regions including cerebrum, cerebellum, midbrain, and thalamus. Samples from cerebrum and cerebellum also showed low OD similar to the negative control. Interestingly, relatively high levels of PrP^Sc were detected in the midbrain and thalamus regions (Table 2). When the same brain regions were tested from cattle with clinical signs, they were strongly positive by EIA. None of the brain regions sampled from the sham-inoculated control or knockout animals inoculated with TME were positive by EIA.

To investigate whether RT-QuIC can detect prion seeding activity from cattle prior to the onset of clinical signs of classical BSE, we compared brainstem homogenates from each of the experimental groups using the RT-QuIC assay with full length bovine rPrP [a.a. 25–241] substrate and the reaction conditions (0.1 mg/ml rPrP final concentration, 300 mM NaCl, and 0.001% SDS) as previously used (29). An increase in ThT fluorescence was observed within 40 h incubation in each quadruplicate reaction seeded with 10⁻⁴ dilution of 10% w/v classical BSE brain homogenate including animal No. 2 and No. 3 (Figure 3). However, fluorescence was not produced in assays seeded with either brainstem of steer No. 1 or negative control animals indicating extremely low or absent prion levels in the brainstem of those animals.

In order to determine whether RT-QuIC can detect PrP^Sc in other brain regions of subclinical cattle, samples from multiple brain regions of a steer No. 1 were assessed. We compared 5 brain regions including brainstem at the level of the obex, cerebrum, cerebellum, midbrain, and thalamus to a negative control sample. As can be seen in Figure 4A, all reactions seeded with the four brain regions (except brainstem) gave strong positive responses within 60 h for the 10⁻² brain dilutions. The reaction seeded with thalamus had the shortest lag time about 25 h. Reactions seeded with cerebrum and cerebellum also produced an increase in ThT fluorescence (Figure 4A) despite having OD values similar to the negative control of the EIA kit confirming that RT-QuIC is more sensitive than the EIA assay for prion detection. For the 10⁻⁴ brain dilution (see Figure 4B), a thalamus sample resulted in ThT fluorescence while assays with other brain regions showed weak or negative ThT responses.

To further compare RT-QuIC reactions of steer No. 1 with those of cattle clinically ill with BSE, we tested brain samples from multiple regions. Table 1 summarizes characteristics of two animals including incubation period, inoculum source, and clinical signs. RT-QuIC detection of prion seeding activity from each brain region from cattle with clinical signs of BSE is demonstrated in Figure 5. RT-QuIC reaction mixtures were seeded with 10⁻² dilutions of brain tissues including brainstem at the level of the obex, cerebrum, cerebellum, midbrain, and thalamus of either animal No. 2 (Figure 5A) or No. 3 (Figure 5B). As can be seen in Figure 5, reactions seeded with brainstem at the level of the obex from animals with clinical signs of BSE had a relatively shorter lag time than other reactions seeded with any other brain sections. Reactions seeded with samples from either the thalamus or the midbrain had slightly longer lag times than reactions seeded with samples from the brainstem. Reactions seeded with samples from the cerebrum and the cerebellum also produced ThT fluorescence with longer lag time than midbrain or thalamus seeded reactions. All reactions seeded with negative brain failed to demonstrate increases in ThT fluorescence.

Samples from steer No. 1 were negative by IHC and WB, but positive in midbrain and thalamus using an EIA method that binds misfolded proteins rather than testing for protease resistance. Therefore, we wanted to test PrP^Sc from the midbrain and thalamus of steer No. 1 for potential PK sensitivity. PK sensitivity testing was performed by measuring the absorbance of bound, misfolded protein before and after treatment with PK using EIA as described above. Brain homogenates of thalamus region from steers No. 1 and No. 2 were prepared in PBS and treated with various concentrations of PK (0–0.5 mg/ml). The results demonstrated that stable absorbance values after treatment with increasing amounts of PK in all samples (Figure 6).

To test whether the ThT fluorescence response of samples from the midbrain and thalamus of steer No. 1 resulted from the residual inoculum, we measured RT-QuIC reactions seeded with brains from knockout (PRNP^−/−) cattle that were intracranially inoculated with TME. We evaluated whether different brain regions of intracranially inoculated knockout animals produced similar RT-QuIC responses to steer No. 1. Prion seeding activity was not detected in any RT-QuIC reactions seeded with brain samples from PRNP knockout cattle, whereas assays with brainstem samples from cattle with clinical signs of BSE or TME demonstrate strong ThT fluorescence (Figure 7).

To allow for comparison of RT-QuIC results with that of bioassay, we inoculated mice with the original classical BSE inoculum or brain homogenate from subclinical steer No. 1. Mice inoculated with 1% classical BSE inoculum (18/18; 100%) developed clinical signs of difficulty moving and poor coordination and were euthanized with a mean incubation period of 299 days. Mice inoculated with 10% brainstem homogenate from steer #1 developed clinical similar signs (16/16; 100%), but with a longer mean incubation period of 363 days despite receiving a more concentrated brain homogenate (Figure 8).