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

Infectivity testing in a reference brain sample and colostrum/milk fractions from scrapie incubating ewes.

<p>(A,B) Survival curve in Tg338 mice (transgenic mice over-expressing ovine VRQ PRP allele) intracerebrally inoculated with colostrum (A) and milk (B), collected from ewes incubating scrapie. Samples were first fractionated into cellular pellet (▵), cream (▿), and casein whey (○). An immunoprecipitation of PrP on magnetic beads coated with anti-PrP antibodies was then carried out. Beads from each fraction were inoculated into five or six Tg338 mice. (A) Colostrum fractions from a ewe harbouring mammary ectopic lymphoid follicles associated with Maedi lesions (white symbols) and from a ewe with a healthy mammary gland (black symbols). (B) Milk fractions from the same ewes as in A (black symbols and white symbols) and of the cellular fraction from a second scrapie incubating ewe with a healthy mammary gland (grey symbols). The experiment was terminated after 900 days (normal Tg338 mouse lifespan). Incubation periods have to be compared to those of successive 1/10 dilutions of brain (obex- vertical dotted lines) material from a sheep clinically affected with scrapie. The start point (neat) corresponds to the inoculation of 2.5 µg of brain tissue per mice. (C) Western-blotting (anti-PrP SHa31 antibody) of without (lane 1) and with (lane 2) PK treatment of brain material from a Tg338 mouse inoculated with scrapie positive brain (10⁻³ diluted); (lanes 2–6) PK digested brain material from mice inoculated with milk and colostrum cellular fraction – (lane 3) milk from a ewe with a healthy mammary gland – (lane 4) colostrum from a ewe with a healthy mammary gland – (lane 5) milk from TSE free control – (lane 6) colostrum from a Maedi affected (ectopic lymphoid follicle) ewe. (D) Intracerebral end point titration of a 12.5% obex homogenate, prepared from a terminally scrapie affected sheep (Langlade isolate), in a Tg338 mouse model. This titration allowed the determination of the infectious dose 50 (ID₅₀) of the brain sample (10^6.8 ID₅₀/g), see the text. (E) Variation of the incubation period as a function of the infectious dose inoculated intracerebrally in Tg338 mice (obex – Langlade isolate), see the text.</p

Immunoprecipitation of PrP in milk and colostrum.

<p>(A) PrP in milk (▵) and colostrum (○), from a negative control animal and three scrapie incubating sheep (casein whey protein extract following NP40/DOC – 10 min at 37°C treatment). PRP levels were measured before (black symbols) and after (white symbols) immunoprecipitation with antibodies (SHa31, SAF-34, and βS-36). The dosage was performed using a two-site sandwich immunoassay (capture antibody 11C6, tracer antibody Bar-224). The positive threshold of the test (0.040 absorbance units) is symbolised by the dotted line. (B–E) PrP contained in different fractions was immunoprecipitated with Sha31/SAF-34/BS36 immunobeads. After washings, PK in PBS (0 to 10 µg in 50 µL) was added to the beads for 10 min at 37°C. Samples were denatured in laemmli's buffer (25 µL), without β-mercaptoethanol, for 5 min at 100°C. Supernatants were then analysed by western blot. (B) 1.4 mL of casein whey, prepared from colostrum (left four lanes) or milk (right four lanes of the gel), from a scrapie incubating ewe (0942 see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000238#ppat-1000238-t004" target="_blank">Table 4</a>), (C) 1.4 mL of casein whey prepared from a TSE free control milk, (D) 100 µl of scrapie positive 2% brain homogenate or (E) 100 µl of scrapie negative 2% brain homogenate.</p

PrP^Sc detection in mammary gland from scrapie-incubating sheep.

<p>(A) PrP^Sc immunolabelling (8G8 monoclonal antibody- DAB brown deposit – bar: 80 µm) in mammary gland from a ewe incubating scrapie (preclinical phase – 15 months old – ARQ/ARQ genotype) and harbouring lympho-proliferative mastitis with ectopic lymphoid follicles (Foll.). In the milk ducts lumen (arrow heads), several PrP^Sc positive cells are identifiable. (B) In mammary gland acini (Aci.), positive PrP^Sc staining can be observed; either associated with cells or distributed as free granules. (C) Double labelling for PrP^Sc (R521 polyclonal serum – black deposits) and CD68 (KiM6 clone – red deposits) indicates that intracellular PrP^Sc in milk ducts and acini lumen is associated with phagocytic cells. (D) PrP^Sc immunolabelling (8G8 anti-PrP antibody – DAB brown deposit- bar: 200 µm) and (E) PET blot (SHa31 antibody – NBT/BCIP black deposits – bar: 200 µm) of two successive mammary gland sections confirmed that material in milk ducts is proteinase K resistant (arrow heads indicate lining).</p

Estimation of infectious titre in colostrum and milk from scrapie incubating ewes with apparently healthy mammary glands or lymphoproliferative mastitis (consecutive to Maedi infection).

<p>For each fraction (cell pellet, casein whey, cream) the quantity of the material submitted to immunoprecipitation process is detailed and linked to the initial volume of colostrum or milk from which it was prepared. In samples for which a 100% attack rate was observed, mean incubation period were used to estimate the infectious titre (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000238#ppat-1000238-g003" target="_blank">Figure 3E</a>). For each considered fraction the infectious titre per ml of starting material was calculated. The global infectious titre per ml of colostrum and milk was finally obtained by adding the value corresponding to each fraction.</p><p>N.A: not available at the moment of writing. *Infectivity was estimated from the only those fractions for which results are available. Consequently the calculated infectious titre/ml of milk is certainly underestimated.</p

PrP^Sc distribution in the organism of scrapie incubating animals.

<p>Groups of 4 ARQ/VRQ and 4 VRQ/VRQ sheep (named a-b-c-d) were killed at different time of the incubation period. Clinical signs occurred at 20 months in VRQ/VRQ animals and at 32 months in ARQ/VRQ. A systematic PrP^Sc detection was realized using immunohistochemistry (8G8 antibody) in a large panel of the collected sheep tissues. PrP^Sc accumulation level was scored according to a semi-quantitative scale: (−) no PrP^Sc, (+) minimal PrP^Sc deposits, (++), (+++) moderate PrP^Sc deposits and (++++) strong PrP^Sc deposits.</p><p><b>LN</b>: Lymph Node; <b>MLN</b>: Mesenteric Lymph Node; <b>PP</b>: Peyer's Patches; <b>ENS</b>: Enteric Nervous System.</p

End-point titration of a brain homogenate (posterior brainstem- 12.5% weight/volume homogenate) in Tg338 mice.

<p>The donor ewe was born and bred in the Langlade Flock. This ewe was at the terminal stage of Scrapie at the moment of culling. Each mouse was intracerebrally inoculated with 20 µl of homogenate. Mice were considered positive when abnormal PrP deposition was detected in brain. Incubation periods are presented as mean+/−SD except for that dilution with which less than 20% of mice were found positive. In that case (*) incubation times of the positive mice are individually presented.</p