Sensitivity of ovRK13 cell assay for the detection of PG127 ovine prion.

<p>A) Morphology of inoculated ovRK13 cultures kept in the same wells during the whole cell assay procedure (d0 is the time of inoculation and d28 is 4 weeks later). B) Sensitivity of ovRK13 cell assay as assessed by immunoblotting. <i>Right panel:</i> Serial 10-fold dilutions (from 10⁻⁴ to 10⁻⁶) of infectious PG127 10% brain homogenate were inoculated to single wells of ovRK13 cells. Four weeks later, inoculated cultures were analyzed for PrP^res by immunoblotting. Positive transmission was detected for dilutions up to 10⁻⁵. No PrP^res was observed when inoculated ovRK13 did not express the ovine PrP (dox-). <i>Left panel:</i> total PrP from infected cells was analyzed before (−) or after (+) PK digestion to illustrate band shift upon PK proteolysis. M are standard molecular mass marker proteins (20, 30 and 40 kDa). C) Sensitivity of ovRK13 cell assay as assessed by Elispot. Replicate wells from the same experiment shown in Fig. 1B were analyzed. Left: representative wells of an Elispot plate showing spots given by ovRK13 cells exposed to the indicated dilutions of PG127. Right: double-logarithmic plot of spot number versus PG127 dilution shown for inoculations in the presence (triangle) or in the absence (square) of dox. For each dilution, the mean value ± SD of 8 measurements is shown. Background values for ovRK13 cells inoculated in the absence of dox are less than 4 spots per 50,000 cells. D) Sensitivity is strongly improved by 2 successive rounds of cell assay. Serial 10-fold dilutions (from 10⁻⁵ to 10⁻⁷) of infectious PG127 10% brain homogenate were inoculated to duplicate wells of ovRK13 cells. Four weeks later, PrP^res in a 1^st set of inoculated cultures was isolated (1^st round) while cultures of the 2^nd set were homogenized to inoculate new ovRK13 cells. Four weeks later, PrP^res was isolated (2^nd round) and all the samples were analyzed by immunoblotting. M are standard molecular mass marker proteins (20, 30 and 40 kDa).</p

Cell assay detection of plastic-bound prions.

<p>Infectivity from 10% PG127 brain homogenate was diluted either in culture medium (prion in culture medium) or in Triton-DOC lysis buffer (prion in detergents) and incubated for 2 h into plastic wells. Samples were removed, wells were thoroughly rinsed and air-dried. OvRK13 cells were then seeded in the presence (+) or in the absence (−) of dox. PrP^res in the cultures was analyzed 4 weeks later by immunoblotting and compared to PrP^res levels in ovRK13 cultures subjected to the standard cell assay (no coating). M are standard molecular mass marker proteins (20, 30 and 40 kDa).</p

Sensitivity of moRK13 cell assay for the detection of RML mouse prions.

<p>Serial 10-fold dilutions (from 10⁻⁴ to 10⁻⁸) of RML 10% brain homogenate were inoculated in duplicate to moRK13 cells and the inoculated cultures were proceeded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020563#pone-0020563-g001" target="_blank">figure 1D</a>. PrP^res was analyzed by western blot after one (A) or two rounds (B) of cell assay. M are standard molecular mass marker proteins in kDa.</p

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

Transfusion of VRQ/VRQ sheep with blood spiked with decreasing amounts of PG127 infected sheep brain homogenate.

<p>200 mL of whole blood was collected from each of 12 TSE Free VRQ/VRQ cheviot sheep. Whole blood pouches were spiked with decreasing amounts of sheep scrapie strain PG127 brain homogenate containing 10^6.6 ID₅₀/g as measured by IC inoculation in tg<i>338</i> mice. Each spiked blood was then transfused back into the sheep of origin. Two sheep were challenged at each dose (indicated as number of ID₅₀ IC in tg338). Sheep were observed until they developed clinical signs or reached 750 days post inoculation when they were euthanized. All recipients were tested for presence of abnormal PrP (PrP^Sc) deposition in brain and various lymphoid tissues by immunohistochemistry. Results confirmed the clinical diagnosis. Incubation periods in recipients are presented as days post inoculation (dpi).</p

Intracerebral inoculation of tg338 mice with VRQ/VRQ sheep whole blood, plasma and red blood cell concentrate.

<p>Blood was collected from three TSE free VRQ/VRQ donor sheep that had been orally challenged with PG127 scrapie (D1, D2, D3) and one TSE free VRQ/VRQ control sheep (C1). The date of collection from the infected animals was 210 days post inoculation. Donors developed clinical scrapie two to five weeks following blood collection. They were euthanized at 227 days, 256 days, 221 days respectively. Whole blood, plasma, red blood cell concentrate (RBC) were each inoculated intracerebrally into 18 tg338 mice (20 µL per mouse). For each component, the volume of whole blood corresponding to the volume inoculated is given. Mice were euthanized when they showed clinical signs of infection or after 250 dpi. Mice were considered infected when abnormal PrP depositions were detected in brain. Infectious titers were estimated using limiting dilution titration method (application of Poisson model) described by Brown et al <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002782#ppat.1002782-Brown3" target="_blank">[23]</a>. Infectious titers are given as the most likely infectious titer and, in parentheses, the values of the lower and upper limits of the 95% confidence interval.</p

Tg338 mice intracerebral inoculation with VRQ/VRQ sheep white blood cells homogenates.

<p>Blood was collected from three TSE free VRQ/VRQ donor sheep that had been orally challenged with PG127 scrapie (D1, D2, D3) and one TSE free VRQ/VRQ control sheep (C1). The date of collection from the infected animals was 210 days post inoculation. Donor sheep developed clinical signs within two to five weeks following blood collection. They were euthanized at 227 days, 256 days, 221 days respectively White blood cells (WBC) were prepared from whole blood and homogenised in 5% glucose solution. Successive 1/10 dilutions of WBC homogenates were inoculated intra-cerebrally to tg<i>338</i> mice (n = 6). The equivalent volume of whole blood inoculated in mice is indicated. Mice were euthanized when they showed clinical signs of infection or after 250 dpi. Mice were considered infected when abnormal PrP depositions were detected in brain. Infectious titer was estimated by the Spearman-Karber method <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002782#ppat.1002782-Markus1" target="_blank">[19]</a>. Infectious titer is expressed as number of ID₅₀ per mL of whole blood. For each samples, the most likely value and (in parentheses) the lower and upper value of the 95% confidence interval are reported.</p

End-point titration in tg338 mice of a 10% brain homogenate and white blood cells samples, collected in VRQ/VRQ sheep orally inoculated with PG127 scrapie.

<p>10% weight/volume homogenate (*) was prepared using posterior brainstem from VRQ/VRQ sheep inoculated with PG127 scrapie isolate and at the terminal stage of disease. Groups of 6 mice that over-express the VRQ ovine PrP (tg<i>338</i>) were intracerebrally (20 µL) inoculated with successive 1/10 dilutions of this homogenate. These data were already used in a previous publication <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002782#ppat.1002782-Andreoletti1" target="_blank">[16]</a>. In parallel, four susceptible VRQ/VRQ sheep (identified as D4, D5, D6 and D7) were orally challenged with PG127 classical scrapie isolate (total dose 10^6.7 ID₅₀ IC in tg338 mice). Scrapie incubation period in sheep were respectively 226 days, 238 days, 228 days and 242 days post inoculation (dpi). Aliquot of the same fresh and PFA 2% fixed WBC than those IV administrated sheep (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002782#ppat-1002782-t005" target="_blank">Table 5</a>) were homogenised in 5% glucose before intracerebral inoculation in tg338 mice (n = 6 per sample, 20 µL per mice). Each mouse received a quantity of WBC that is equivalent to 2.5 mL of starting whole blood. Mice were observed till occurrence of clinical signs compatible with a transmissible spongiform encephalopathy and considered positive when abnormal PrP deposition was detected in brain. Incubation periods (days post inoculation: dpi) in mice are presented as mean +/−SD except for those dilutions with which less than 50% of mice were found positive. In that case individual incubation period are reported (†). Infectious titers were estimated by the Spearman-Karber method <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002782#ppat.1002782-Markus1" target="_blank">[19]</a>. Infectious titer is expressed as number of ID₅₀ per mL of whole blood. For each samples, the most likely value and, in parentheses, the lower and upper value of the 95% confidence interval are reported.</p

Intravenous administration in sheep and intracerebral challenge in tg338 mice of blood derived products prepared from PG127 scrapie infected sheep.

<p>Four scrapie-susceptible VRQ/VRQ sheep (identified as D4, D5, D6 and D7) were orally challenged with 1 g of sheep scrapie strain PG127 infected brain homogenate containing 10^6.7 IC ID₅₀/g as measured by IC inoculation of tg338 mice Scrapie incubation periods were 226 dpi, 238 dpi, 228 dpi and 242 dpi, respectively. Blood was collected at 217 dpi, <i>i.e</i> few days (D4) to three weeks (D7) before clinical onset. Plasma and white blood cells (WBC) were prepared from 500 mL of whole blood. Half of the WBC preparation from each animal was fixed with paraformaldehyde (PFA 2% final concentration). Whole Blood, plasma and both fresh and fixed WBCs (re-suspended in 5% glucose), each corresponding to 200 mL of whole blood, were administered intravenously to VRQ/VRQ TSE free recipients. In addition, Plasma volume equivalent to 20 mL of whole blood was also intravenously administered to sheep. Recipients were euthanized when symptomatic with scrapie. 450 dpi (or 380 dpi for the 20 mL plasma) recipients that were still alive and apparently healthy were euthanized. Incubation periods in recipients are presented in days post inoculation (dpi). All recipient sheep were tested for the presence of abnormal PrP deposition in brain and various lymphoid tissues by immunohistochemistry.NA: not assessed.</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