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

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

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

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

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

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

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

PrP^Sc in central nervous system, lympho-reticular system, mammary gland and milk duct lumen of natural scrapie exposed ewes bearing various genotypes at codons 136, 154 and 171 of the PRP gene.

<p>All investigated animals (n = 175) were PCR positive for Maedi lentivirus. Abnormal PrP (PrP^Sc) detection was carried out using Western Blotting (SHa31 anti-PrP antibody), ELISA (TeSeE Sheep and Goat, Bio-Rad) and immunohistochemistry (8G8 anti-PrP antibody). The majority of susceptible genotype sheep (ARQ/VRQ- VRQ/VRQ and ARQ/ARQ) were clinical suspect for scrapie at culling.</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

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