PMCA analysis of white blood cells and platelets samples.

<p>Platelets (A) and white blood cells (WBC) (B) from sheep D2 collected at the indicated time points (dpi) were subjected to two successive rounds of PMCA. Unseeded reactions were run in parallel. Samples were processed for PrP^res isolation and analyzed by immunoblotting. A western-blotting positive control (cont) is included in each gel.</p

Cell-based assay of white blood cells infectivity from asymptomatic scrapie sheep.

<p>White blood cells from 5 infected sheep (D1 to D5) were isolated 80 days and 130 days post inoculation (dpi) when sheep were still asymptomatic. White blood cell homogenates (4×10⁷ cells) were inoculated to recipient ovRK13 cells. After 2 successive rounds of cell assay, the cultures were assayed for PrP^res by immunoblotting. PrP^res level is higher in cells infected with D3 130 dpi sample but its banding pattern is similar to that in cells infected with the other samples. M are molecular mass marker proteins (20, 30 and 40 kDa).</p

Evaluation of the infectivity present in platelets prepared from scrapie infected sheep by two different methods: PMCA and inoculation into tg338 mice (bioassay).

<p>Five susceptible VRQ/VRQ sheep were orally challenged with 2 g of brain homogenate (10^6.6 ID₅₀/g IC in tg<i>338</i> mice) between 6 and 10 months of age. The five VRQ/VRQ sheep respectively died at 198, 193, 198, 194 and 191days post inoculation (dpi). Classical scrapie was confirmed by histopathology (vacuolar change in central nervous system) and detection of abnormal PrP deposit in central nervous system and lymphoid tissues. At different time points, whole blood was collected from each donor and aliquots of platelets corresponding to 15 mL of plasma were prepared. Platelet homogenates (in 200μL) were inoculated in groups of six tg338 mice. Mice were monitored up to occurrence of TSE compatible clinical sign onset or killed at 250 days post inoculation. All mice were tested for presence of abnormal PrP deposition in brain. Incubation period in mice are presented in days (+/−SD). When less than 3 mice were positive, individual incubation period are given. In parallel the same homogenates were tested by PMCA (using tg338 mice brain homogenate as substrate). Each sample was run in 5 replicates for 2 successive rounds and the number of positive reactions is presented.</p

Evaluation of the infectivity present in white blood cells prepared from scrapie infected sheep by Cerebellar Organotypic Slice Culture Assay.

<p>Immunoblots of PK-treated slice culture homogenates probed with anti-PrP antibody Sha31, showing PrP^res accumulation in slice culture. (A) Cerebellar organotypic slices were prepared from tg338 pups and maintained in culture during 42 days <i>in vitro</i> after exposure to white blood cells prepared from blood collected from five scrapie infected sheep (D1, D2, D3, D4 and D5) at different times: 50 days post inoculation (dpi), 80 dpi, 130 dpi and at the terminal stage (180 dpi). For quantification purposes, slice cultures were also exposed to serial dilutions of PG127 scrapie-infected brain stock prepared from terminally ill tg338 mice, previously used <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104287#pone.0104287-ArellanoAnaya1" target="_blank">[31]</a>. To visualize low levels of PrP^res, membranes were exposed over-night (B).</p

Detection of PrP^res in peripheral tissue in pigs with clinical disease induced by intracerebral challenge with sheep-passaged bovine spongiform encephalopathy agent

<div><p>Bovine spongiform encephalopathy (BSE) can be efficiently transmitted to pigs via intracerebral inoculation. A clear link has been established between the consumption of products of bovine origin contaminated with the BSE agent and the development of variant Creutzfeldt-Jakob disease in humans. Small ruminants can also naturally develop BSE, and sheep-adapted BSE (Sh-BSE) propagates more efficiently than cattle BSE in pigs and in mouse models expressing porcine prion protein. In addition, Sh-BSE shows greater efficiency of transmission to human models than original cow BSE. While infectivity and/or abnormal PrP accumulation have been reported in the central nervous system in BSE-infected pigs, the ability of the agent to replicate in peripheral tissues has not been fully investigated. We previously characterized the presence of prions in a panel of tissues collected at the clinical stage of disease from pigs experimentally infected with Sh-BSE. Western blot revealed low levels of PrP^res accumulation in lymphoid tissues, nerves, and skeletal muscles from 4 of the 5 animals analysed. Using protein misfolding cyclic amplification (PMCA), which we found to be 6 log fold more sensitive than direct WB for the detection of pig BSE, we confirmed the presence of the Sh-BSE agent in lymphoid organs, nerves, ileum, and striated muscles from all 5 inoculated pigs. Surprisingly, PrP^res positivity was also detected in white blood cells from one pig using this method. The presence of infectivity in lymphoid tissues, striated muscles, and peripheral nerves was confirmed by bioassay in bovine PrP transgenic mice. These results demonstrate the ability of BSE-derived agents to replicate efficiently in various peripheral tissues in pigs. Although no prion transmission has been reported in pigs following oral BSE challenge, our data support the continuation of the Feed Ban measure implemented to prevent entry of the BSE agent into the feed chain.</p></div

PrP^res detection in PMCA reactions seeded with tissue samples collected from a Sh-BSE clinically affected pig.

<p>Tissues: brain, sciatic nerve, brachial nerve, mesenteric lymph node, oculomotor muscle, ileum, spleen, and white blood cells (WBC). The animal was culled at 30 months after intracerebral inoculation with Sh-BSE. After 3 rounds of PMCA (48 hours each), PrP^res amplification was detected at dilutions of 10⁻¹ (a) and 10⁻² (b) in all the tissues analysed. No PrP^res was amplified in the mesenteric lymph node, ileum, or spleen at a dilution of 10⁻³ (c). Results obtained for 10⁻¹ dilution after 3 rounds of PMCA in reactions seeded with tissues from a negative control pig (d). Control: PK digested classical scrapie isolate.</p

Relative performance of protein misfolding cyclic amplification of porcine Sh-BSE using brain tissue from ARQ/ARQ ovine PrP transgenic mice as substrate.

<p>PMCA reactions were seeded with a 10-fold dilution series (10⁻¹–10⁻¹¹) of 8 different tissues collected from 5 Sh-BSE-infected pigs. For each tissue, the last PrP^res-positive dilution as detected by Western blot after 1 (white), 2 (grey), and 3 (black) consecutive amplification rounds (96 cycles in a Misonix 4000 sonicator) is shown. The following tissues were assayed: brain, sciatic nerve, brachial nerve, mesenteric lymph node, extraocular muscle, ileum, spleen, and white blood cells.</p

Intracerebral inoculation of tgBov mice with a panel of tissues collected from a Sh-BSE clinically affected pig.

<p>Intracerebral inoculation of tgBov mice with a panel of tissues collected from a Sh-BSE clinically affected pig.</p

Western-blot analysis to detect the proteinase K-resistant core fragment (PrP^res) of the pathologic prion in the brain of 5 pigs experimentally infected with Sh-BSE.

<p>PrP^res western-blot profile from the original Sh-BSE isolate, characterized by predominance of the diglycosylated band, is shown for comparison. Immunodetection was performed using the monoclonal Sha31 antibody. A PK digested classical scrapie isolate (Dawson strain) was used as positive control (Control).</p

Western blot detection of PrP^res in the original inocula prepared with tissues collected from Sh-BSE-inoculated pigs with clinical disease.

<p>Western blot detection of PrP^res in the original inocula prepared with tissues collected from Sh-BSE-inoculated pigs with clinical disease.</p