Individual sheep results of PrP^Sc detection by IHC/IDEXX EIA in each peripheral organ studied.

<p>The OD value obtained by IDEXX EIA is indicated for each sample.</p><p>NT, no tested. NC, no conclusive results.</p

Distribution of Peripheral PrP<i>^Sc</i> in Sheep with Naturally Acquired Scrapie

<div><p>Accumulation of prion protein (PrPSc) in the central nervous system is the hallmark of transmissible spongiform encephalopathies. However, in some of these diseases such as scrapie or chronic wasting disease, the PrPSc can also accumulate in other tissues, particularly in the lymphoreticular system. In recent years, PrPSc in organs other than nervous and lymphoid have been described, suggesting that distribution of this protein in affected individuals may be much larger than previously thought. In the present study, 11 non-nervous/non-lymphoid organs from 16 naturally scrapie infected sheep in advanced stages of the disease were examined for the presence of PrPSc. Fourteen infected sheep were of the ARQ/ARQ PRNP genotype and 2 of the VRQ/VRQ, where the letters A, R, Q, and V represent the codes for amino-acids alanine, arginine, glutamine and valine, respectively. Adrenal gland, pancreas, heart, skin, urinary bladder and mammary gland were positive for PrPSc by immunohistochemistry and IDEXX HerdChek scrapie/BSE Antigen EIA Test in at least one animal. Lung, liver, kidney and skeletal muscle exhibited PrPSc deposits by immunohistochemistry only. To our knowledge, this is the first report regarding the presence of PrPSc in the heart, pancreas and urinary bladder in naturally acquired scrapie infections. In some other organs examined, in which PrPSc had been previously detected, PrPSc immunolabeling was observed to be associated with new structures within those organs. The results of the present study illustrate a wide dissemination of PrPSc in both ARQ/ARQ and VRQ/VRQ infected sheep, even when the involvement of the lymphoreticular system is scarce or absent, thus highlighting the role of the peripheral nervous system in the spread of PrPSc.</p></div

Details of the scrapie infected sheep used in the study.

<p>From each animal the identification number, age, flock of origin (outbreak), genotype and immunohistochemical and IDEXX EIA test results from brainstem and spleen are included.</p>1<p>Age was determined by the dental status of the animals.</p>2<p>PrP^Sc presence and score: positive (+) or negative (−) for IHC.</p>3<p>Limit detection of PrP^Sc by IDEXX EIA test.</p

Immunohistochemical detection of PrP^Sc in pancreas using L42 PrP antibody.

<p>A–B) Pancreas from naturally scrapie-infected sheep. PrP^Sc immunolabeling was detected only in relation to intrapancreatic ganglia neurons, showing a granular intracytoplasmic (A; x20) or a perineuronal (B; x40) labelling. C) Pancreas from an uninfected control sheep in which an intrapancreatic ganglion is indicated by the dotted line (x40). No PrP^Sc immunolabeling is present.</p

Immunohistochemical detection of PrP^Sc in skin.

<p>A–B) Skin from a naturally scrapie-infected sheep with PrP^Sc immunolabeling located mainly at the epidermis. PrP^Sc was detected using L42 (A; x40) and F89 (B; x40) PrP monoclonal antibodies. C) Skin from an uninfected control sheep in which no PrP^Sc immunolabeling is present (x40).</p

Immunohistochemical detection of PrP^Sc in heart and skeletal muscle using L42 PrP antibody.

<p>A) Heart from a naturally scrapie-infected sheep. PrP^Sc immunolabeling is located among myocardial myocytes (x63). B) Heart from an uninfected control sheep in which no PrP^Sc immunolabeling is present (x40). C) Cross section of a nerve fiber bundle located within a skeletal muscle sample from a naturally scrapie-infected sheep (x63). A periaxonal PrP^Sc immunolabeling can be observed in some nerve fibers (arrows). D) Skeletal muscle from a naturally scrapie-infected sheep in which PrP^Sc immunolabeling is associated with a neuromuscular spindle (cross section; x63). The neuromuscular spindle was identified by its typical structure: small groups of specialized striated muscle fibers (asterisks), which are thinner than regular muscle fibers, surrounded by a capsule of connective tissue (arows). E) Skeletal muscle from an uninfected control sheep in which no PrP^Sc immunolabeling is present (x10). A detailed image of a nerve fiber bundle immersed in the muscle is shown in the insert picture.</p

Immunohistochemical detection of PrP^Sc in mammary gland using L42 PrP antibody.

<p>A) Mammary gland from a sheep simultaneously infected by scrapie and by Maedi-visna infection causing a mastitis (x10). PrP^Sc immunolabeling is located within the lymphofollicular inflammatory lesions. In the mammary gland acini (arrows) next to the inflammatory lesions no PrP^Sc immunolabeling can be observed. B) Mammary gland from an uninfected control sheep with mastitis associated with Maedi-visna infection in which no PrP^Sc immunolabeling is present (x10). C) Mammary gland from a naturally scrapie-infected sheep (x10). PrP^Sc immunolabeling can be observed in the nerves immersed in the interlobular connective tissue (x10). A detailed image of a nerve fibers bundle in which a periaxonal PrP^Sc immunolabeling can be observed (arrows) is shown in the insert picture. D) Mammary gland from an uninfected control sheep in which no PrP^Sc immunolabeling is present (x20). Cross section of a nerve fiber is indicated by an arrow.</p

Immunohistochemical detection of PrP^Sc in urinary bladder and kidney using L42 PrP antibody.

<p>A) Urinary bladder muscularis from a naturally scrapie-infected sheep (x40). There is a weak PrP^Sc immunolabeling located in the connective tissue adjacent to the smooth muscle bundles, probably associated with vasculonervous elements. B) Urinary bladder muscularis from an uninfected control sheep in which no PrP^Sc immunolabeling is present (x20). The connective tissue (indicated by arrows head) with vasculonervous elements located between the smooth muscle can be clearly seen with low magnification. C) Kidney from a naturally scrapie-infected sheep. PrP^Sc immunolabeling is observed mainly at the vascular pole of a renal corpuscle (x40). D) A renal corpuscule of a kidney from an uninfected control sheep in which no PrP^Sc immunolabeling is present (x20).</p

PNGase digestion of vCJD-infected Tg340 and vCJD-PMCA-TgNN6h mice brains.

Samples were digested with 170 μg/mL PK and 3 ng/μL PNGase F, loaded on SDS-PAGE and subjected to Western Blot using monoclonal antibody 3F4 (1:10,000). Note that PNGase treatment reduced vCJD-infected Tg340 three-band pattern to a single band of approximately 19 kDa, but did not affect the banding pattern of vCJD-PMCA-infected TgNN6h, which in addition retained the ~15-kDa band that we have previously observed in this unglycosylated model, assumed to be an endogenous proteolytic fragment. PNGase digestion did not cause the emergence of the 15-kDa band in Tg340 samples, indicating that it is not the unglycosylated form of a fragment present in Tg340 brains. (DOCX)</p

Recovery of BSE banding pattern on passage from TgNN6h to BoTg110 of BSE, sBSE and pBSE isolates.

Brain homogenates at 1% from challenged mice were digested with 85 μg/mL (for original isolates) or 170 μg/mL (for TgNN6h and BoTg110 brains) of proteinase K and analyzed by Western blot using 6H4 antibody (1:10,000). A transition from a classical three-banded BSE pattern in the original isolates to a single 19-kDa band-containing pattern in TgNN6h mice was observed, followed by a complete recovery of the three-banded pattern in BoTg110 mice. Note that the banding pattern of the pBSE original isolate differs from the classical BSE pattern in being predominantly monoglycosylated (a hallmark imposed by porcine PrPC), while after passage to BoTg110 the pattern is identical to those of other BSE sources. Red arrows indicate passage history.</p