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

Activation of microglia in retinas of cattle inoculated with BSE.

<p>An antibody directed against Iba-1 was used to label microglia in retinal sections. Microglia in retinal sections from sham inoculated and negative control cattle had a stratified appearance with processes primarily in the outer plexiform and inner plexiform layers (Fig. 5A). In sections from cattle inoculated with classical BSE, the microglia appeared to be more amoeboid in morphology (Fig. 5B) but not more numerous than was observed in negative controls. In contrast, retinal sections from cattle inoculated with BSE-H had more numerous microglia with an amoeboid morphology (Fig. 5C). The ages of the animals in the figure are 6.8, 5.9 and 5.7 years in A, B and C respectively. Abbreviations: OFL = optic fiber layer, GCL = ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer; OPL = outer plexiform layer; ONL = outer nuclear layer; OS = outer segments. Scale bars = 20uM</p

Changes in retinal thickness over time in animals inoculated with classical BSE and US BSE-H.

<p>Total retinal thickness was measured over time using optical coherence tomography. Animals were compared to their baseline values in 6, 12 and 15 months post inoculation (MPI), and compared to the control cohort at 12 MPI. Values in parenthesis the standard error of the mean, values in itallics in are the sample size, significant changes are indicated by shading.</p><p>* = p < 0.05;</p><p>** = p< 0.01;</p><p>*** = p< 0.0001.</p><p>Changes in retinal thickness over time in animals inoculated with classical BSE and US BSE-H.</p

Changes in the electroretinograms over time in animals inoculated with classical BSE.

<p>B-wave amplitude and implicit time in dark-adapted animals with either dim (-20 dB) or bright (0 dB) flash were analyzed over time. Sample size for each time point are in italics. Values in parenthesis the standard error of the mean. MPI = months post inculcation.</p><p>* = p < 0.05;</p><p>** = p< 0.01.</p><p>Changes in the electroretinograms over time in animals inoculated with classical BSE.</p

Müller glia are activated in the retinas of cattle inoculated with BSE.

<p>An antibody directed against glial fibrillary acidic protein (GFAP) was used to label Müller glia in retinal sections. Retinas from sham inoculated and negative control cattle had GFAP-immunoreactivity only in the optic fiber layer (asterisk), in astrocytes, and endfeet of Müller glia (Fig. 4A). Retinal sections from cattle inoculated with classical BSE had increased immunoreactivity in the optic fiber layer and in occasional thin processes of Müller glia spanning the inner plexiform layer (arrows; Fig. 4B). Sections from cattle inoculated with BSE-H (Fig. 4C) had robust immunoreactivity in the optic fiber layer and consistently in Müller glial processes (arrow), which appeared hypertrophied compared to sections from animals inoculated with classical BSE. The ages of the animals in the figure are 2.7, 6.4 and 5.7 years in A, B and C respectively. Abbreviations: OFL = optic fiber layer, GCL = ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer; OPL = outer plexiform layer; ONL = outer nuclear layer; OS = outer segments. Scale bars = 50 μM.</p