RT-QuIC sodium chloride titration for TME-infected and uninfected cattle brain samples.

<p>RT-QuIC reactions were seeded with 10⁻⁴ (A, B) and 10⁻⁶ dilution (C, D) of TME-infected and uninfected cattle brain homogenates using a range of NaCl concentrations (100–500 mM) with the full-length bPrP wild type (aa 25–241) as substrate. Data are presented as mean ThT fluorescence of 8 reactions conducted as 2 repeats of 4 reactions. The positive threshold was calculate as ~10,000 relative fluorescence units of normal cattle brain homogenates.</p

Use of bovine recombinant prion protein and real-time quaking-induced conversion to detect cattle transmissible mink encephalopathy prions and discriminate classical and atypical L- and H-Type bovine spongiform encephalopathy - Fig 6

<p><b>RT-QuIC detection of C-, L-, and H-BSE prion seeding activity using bovine prion proteins (25–241) wild type (A) and its disease associated form E211K protein (B).</b> RT-QuIC reaction mixtures were seeded with 10⁻⁴ dilutions of brain tissues from uninfected, C-BSE-affected (cyan and grey lines), L-BSE-affected (red and black lines) and H-BSE-affected (pink, blue, yellow, green lines) cattle. A final SDS concentration of 0.001% in combination with 300 mM NaCl was used with the substrates. Data are presented as mean ThT fluorescence of 8 reactions conducted as 2 repeats of 4 reactions. The positive threshold was calculate as ~10,000 relative fluorescence units of normal cattle brain homogenates.</p

Animal donor information.

<p>Brain tissues collected from cattle that were normal or clinically ill with BSE were used to prepare homogenates.</p

Western blot of a representative brain sample of each BSE strain and TME in cattle.

<p>Brainstem samples were characterized by western blotting (mAb 6H4). Lanes from left: 1. French L-type (#6895, 2 mg);; 2. U.S. H-type (#80, 2 mg); 3. Classical (#6836, 1 mg); 4.Negative Control (#6969, 2 mg); 5. Biotinylated protein marker; 6. Bovine TME (Animal #52AA, 2 mg); 7. Negative Control (#6969, 2mg); 8. Biotinylated protein marker.</p

RT-QuIC sodium chloride titration for different types of BSE-infected cattle brain samples.

<p>RT-QuIC reactions were seeded with 10⁻⁴ and 10⁻⁶ dilution of C-, L, and H-BSE-infected cattle brain homogenates using a range of NaCl concentrations (100–500 mM) with the full-length bPrP wild type (aa 25–241) as substrate. Data are presented as mean ThT fluorescence of 8 reactions conducted as 2 repeats of 4 reactions. The positive threshold was calculate as ~10,000 relative fluorescence units of normal cattle brain homogenates.</p

RT-QuIC sodium chloride titration for TME-infected and uninfected cattle brain samples.

<p>RT-QuIC reactions were seeded with 10⁻⁴ (A, B) and 10⁻⁶ dilution (C, D) of TME-infected and uninfected cattle brain homogenates using a range of NaCl concentrations (100–500 mM) with the full-length bPrP E211K protein (aa 25–241) as substrate. Data are presented as mean ThT fluorescence of 8 reactions conducted as 2 repeats of 4 reactions. The positive threshold was calculate as ~10,000 relative fluorescence units of normal cattle brain homogenates.</p

Results of antemortem ophthalmologic testing.

<p>(<b>A</b>)Comparison of mean ERG b-wave implicit time at 0, 6, and 9 months post-inoculation (MPI). Implicit time was prolonged at 9 MPI compared to baseline (0 MPI) and 6 MPI values for all test conditions. Test 1, dark adapted 0.024 cd•s/m²; test 2, dark adapted 2.45 cd•s/m²; test 3, light adapted 2.45 cd•s/m². (<b>B</b>) Optical Coherence Tomography (OCT) was used to measure full retinal thickness, (including all retinal layers) of Calf 83 prior to inoculation (0 MPI), nine-months post inoculation (9 MPI) and one day prior to necropsy (approximately 9.8 MPI). There was an appreciable decrease in retinal thickness from 0 MPI to 9 MPI. Retinal thickness was also decreased relative to an age-matched, genotype matched, non-inoculated control (Calf 78). Error bars are standard deviation based on 10 measurements. Abbreviations: cd•s/m² = candela seconds per meter squared; msec = milliseconds.</p

Western blot analysis with a panel of monoclonal antibodies.

<p>(<b>A</b>) Samples from five brain regions of calf #83 demonstrate the characteristic 3-band profile of PrP^Sc when developed with mAb 6H4. The unglycosylated, monoglycosylated, and diglycosylated bands are of similar intensity. Note the presence of an additional band at approximately 23 kDa in the sample from cerebellum. (<b>B</b>) Western blot analysis with monoclonal antibody P4 showing the characteristic 3 band profile of PrP^Sc for both E211K and BSE-H, but an absence of detectable PrP^Sc for classical BSE. (<b>C</b>) Western blot analysis with mAb SAF-84 showing comparison of PrP^Sc profiles in brain of classical, E211K, and H-type BSE. E211K and H-type BSE have a 4^th band at 14 kDa, whereas classical BSE does not. (<b>D</b>) All BSE-H samples appear similar after PNGase F treatment (mAb 6H4). Samples were loaded at 0.6–0.8 mg (A–C) or 0.1–0.3 mg (D) equivalents of brain tissue per lane. Molecular weight standards flank the blot and the molecular weight in kDa is indicated to the left of the blot.</p

Retinal pathology corroborates antemortem findings.

<p>(<b>A</b>) PrP^Sc immunoreactivity demonstrates that abundant PrP^Sc (red) accumulates in the retina. Original magnification 20×. (<b>B</b>) GFAP immunoreactivity (brown) in the Müller glia (arrow) indicates a response to retinal injury. Original magnification 20×.</p

Spongiform change was present at all levels of the brain examined.

<p>(<b>A, B</b>) Vacuoles were numerous and evenly spread in the piriform cortex. Original magnification 4× and 10×, respectively. (<b>C, D</b>) The parasympathetic nucleus of the vagus nerve contained few but definitive spongiform lesions. Vacuoles occur predominantly in the neuropil rather than perikarya. Original magnification 2× and 40×, respectively.</p