PrP^Sc detected by immunohistochemistry in scrapie-affected Sarda sheep.

<p>(A) Immunohistochemical analysis of the cortex, medulla and papillae of naturally scrapie-sick sheep and control sheep derived from scrapie-free flocks. Antibodies 2G11 and F99 were used. Scale bar = 30 µm. (B) Immunofluorescence using 2G11 identifies tubular deposits in the medullar-cortical junction of the kidney of a scrapie-sick sheep. Scrapie-free control is devoid of detectable positive signal.</p

Number (N), age, PrP genotype and clinical status of the sheep included in the study.

<p>Sheep are grouped as scrapie-affected sheep or negative control. The number (N) of the flocks from which the sheep were selected is also indicated.</p>*<p>Sheep from scrapie-affected flock</p

Quantification of PrP^Sc in renal homogenates of naturally scrapie-sick sheep.

<p>(A) Conventional Western blot analysis of various brain dilutions after proteinase K (PK) digestion. Undigested brain is loaded to control for PK digest (lane 1). Kidney homogenates from independent scrapie sick animals were loaded (lanes 7–10). (B) Relative quantification of PrP^Sc signal intensity found in kidney homogenates in relation to brain derived PrP^Sc. Our results indicate that the highest amounts of renal PrP^Sc detected were appr. up to 20% of the PrP^Sc signal detected in brain.</p

Additional file 1 of Advances in understanding the genetic architecture of antibody response to paratuberculosis in sheep by heritability estimate and LDLA mapping analyses and investigation of candidate regions using sequence-based data

Additional file 1: Figure S1. Quantile–quantile (Q–Q) plot corresponding to the LDLA mapping that was carried out based on CAR. The plot contains the observed − log10(p-value) obtained by the LDLA analysis (y axis) plotted against the expected − log10(p-value) (x axis). Figure S2. Quantile–quantile (Q–Q) plot corresponding to the LDLA mapping that was carried out based on SP. The plot contains the observed − log10(p-value) obtained by the LDLA analysis (y axis) plotted against the expected − log10(P-value) (x axis)

Number (N) of examined Sarda breed sheep, with or without clinical signs, displaying PrP^Sc in the kidney, indicated as percentage (%).

<p>The results are only referred to conventional Western blot examinations.</p

PET blot analysis identifying PrP^Sc in the tubular structures of the cortex and medulla in naturally scrapie-sick Sarda sheep.

<p>(A) Brains of scrapie-sick (left) and healthy sheep (right) as a control for PET blot analysis procedure. Scale bar = 100 µm. (B) Low magnification of transverse paraffin sections from kidneys of a naturally scrapie-sick and a healthy sheep demonstrating PrP^Sc in the medullary and cortical regions of the kidney. Dotted lines indicate the locus of the kidney capsule or the edge of the tissue section. The small transparent box indicates the region shown at higher magnification and resolution in figure C. Scale bar = 1 cm. (C) PrP^Sc deposits and tubular structure fit together in the cortical part of the kidney in scrapie-sick sheep. Scale bar = 100 µm. (D) PrP^Sc is also found to a high degree in the collecting ducts of the medulla (arrowhead) as well as in the papillae (arrow) of kidneys derived from naturally scrapie infected sheep. Scale bar = 30 µm.</p

Western Blotting PrP^Sc deposition in scrapie-affected Sarda sheep.

<p>(A) Detection of PrP^Sc by conventional Western blot in the cortical and medullar part of kidneys. Brain and kidney homogenates were treated with (+) or without (−) proteinase K (PK), amounts of kidney and brain represented in each line are 1mg and 1g respectively. S.b. = brain derived from a naturally scrapie-sick sheep; h.b. = brain derived from a scrapie-free sheep control; h.k. = kidney derived from a scrapie-free sheep control, s.k. = kidney derived from a naturally scrapie-sick sheep. kD = kilo Dalton, M = medulla, C = cortex. (B) Detection of PrP^Sc by NaPTA Western blot in kidney homogenate derived from different Sarda sheep. Substantial individual variation of the Western blotting signal was observed among the sheep examined. Spike control = brain homogenate derived from a scrapie–sick sheep spiked into negative control kidney homogenate. h.b. = brain derived from a scrapie-free sheep control. 2000–4000 µg of renal homogenate were used as a starting material to perform NaPTA precipitation.</p

PrP^Sc is detected only in inflamed sheep mammary glands containing newly formed lymphoid follicles.

<p>Inflammatory cell immunophenotyping in serial paraffin-embedded sections of mammary glands from sheep simultaneously affected by scrapie and lymphofollicular, lymphoproliferative/non-lymphofollicular, or granulomatous mastitis. A normal ovine palatine tonsil is also shown for comparison. CNA-42, CD3, and CD79 were used as markers for FDCs, T cells, and B cells (Panel A), while CD172a, CD68, and CD11c were used for activated macrophages and DCs (Panel B), respectively. In panel C, a representative set of negative controls are shown. These were obtained by omitting all the primary antibodies used on the tonsil sections (photos 1, 2, 3, 4, 6, and 7), as well as from granulomatous mastitis (photo 5). The omitted primary antibodies are indicated on each photo. It is noteworthy that the relative positioning of these cell populations was similar in newly formed lymphoid follicles and constitutive follicles normally hosted inside the palatine tonsil. CD172a⁺ macrophages were the most representative cell population in granulomatous mastitis, which also displayed a moderate presence of T and B cells and DCs. CD3⁺ T cells were the most abundant cellular component in the context of lymphoproliferative/non-lymphofollicular mastitis. PrP^Sc deposition occurred in co-localization with CD79⁺ B cells and the CNA42⁺ FDC network both in palatine tonsil and in newly formed lymphoid follicles. Immune reactions were visualized by 3-3′-diaminobenzidine (DAB) chromogen, with F99 primary MoAb being used for PrP^Sc detection. Mayer’s hematoxylin counterstain. Scale bar = 100 µm.</p

Inflammatory lesions patterns in ovine mammary glands.

<p>Micrographs show the inflammatory lesions’ morphology in mammary glands from sheep concurrently affected by scrapie and mastitis. Representative patterns of lymphofollicular (A), granulomatous (B), and lymphoproliferative/non-lymphofollicular (C) mastitis. A histologically normal ovine mammary gland is also shown (D). Hematoxylin-eosin (H&E) stain. Scale bar = 100 µm.</p

Details of the 27 sheep with clinical scrapie and chronic inflammation affecting a number of organs.

<p>Results of PrP^Sc IHC at the sites of inflammation are also shown. *PrP^Sc deposition was seen occurring only within newly formed lymphoid follicles, either adjacent or not to granulomatous inflammatory lesions.</p