Correlation between infectivity and disease associated prion protein in the nervous system and selected edible tissues of naturally affected scrapie sheep

<div><p>The transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of fatal neurodegenerative disorders characterised by the accumulation of a pathological form of a host protein known as prion protein (PrP). The validation of abnormal PrP detection techniques is fundamental to allow the use of high-throughput laboratory based tests, avoiding the limitations of bioassays. We used scrapie, a prototype TSE, to examine the relationship between infectivity and laboratory based diagnostic tools. The data may help to optimise strategies to prevent exposure of humans to small ruminant TSE material via the food chain. Abnormal PrP distribution/accumulation was assessed by immunohistochemistry (IHC), Western blot (WB) and ELISA in samples from four animals. In addition, infectivity was detected using a sensitive bank vole bioassay with selected samples from two of the four sheep and protein misfolding cyclic amplification using bank vole brain as substrate (vPMCA) was also carried out in selected samples from one animal. Lymph nodes, oculomotor muscles, sciatic nerve and kidney were positive by IHC, WB and ELISA, although at levels 100–1000 fold lower than the brain, and contained detectable infectivity by bioassay. Tissues not infectious by bioassay were also negative by all laboratory tests including PMCA. Although discrepancies were observed in tissues with very low levels of abnormal PrP, there was an overall good correlation between IHC, WB, ELISA and bioassay results. Most importantly, there was a good correlation between the detection of abnormal PrP in tissues using laboratory tests and the levels of infectivity even when the titre was low. These findings provide useful information for risk modellers and represent a first step toward the validation of laboratory tests used to quantify prion infectivity, which would greatly aid TSE risk assessment policies.</p></div

Proportion of sheep testing PrP^Sc positive in each tissue by three Western blot methods.

<p>Number in parentheses is the mean relative intensity (%) compared with 1/8 diluted brain sample.</p><p>Proportion of sheep testing PrP^Sc positive in each tissue by three Western blot methods.</p

Bioassay of edible tissues in voles.

<p>* Clinically healthy voles culled at the end of the experiment.</p><p>Bioassay of edible tissues in voles.</p

Comparison of PrP^Sc, infectivity and converting activity by PMCA in the inocula.

<p>* For the brain dilutions, only positive and negative dilutions are indicated. For the edible tissues a semi-quantification is provided. The WB signal was semi-quantified as follows:</p><p>+++ = a tissue positive in all experiments and with a WB signal between brain dilutions −2 and −3</p><p>++ = a tissue positive in at least 2/3 experiments and with a WB signal similar to brain dilution −3 or less</p><p>+/− = a tissue positive in 1/3 experiments and with a WB signal below brain dilution −3</p><p>** For the brain dilutions, only positive and negative dilutions are indicated. For the edible tissues a semi-quantification is provided. The bioassay results were categorised as follows (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122785#pone.0122785.t005" target="_blank">Table 5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122785#pone.0122785.s002" target="_blank">S2 Fig.</a>)</p><p>+++ = similar to brain dilution −3 or more efficient</p><p>++ = between brain dilutions −3 and −4</p><p>+ = similar to brain dilution −4 or less efficient</p><p>Comparison of PrP^Sc, infectivity and converting activity by PMCA in the inocula.</p

Different levels of abnormal PrP immunolabelling in selected tissues from scrapie positive sheep using R145 as primary antibody and Mayer’s haematoxylin as counterstain.

<p>A: Widespread abnormal PrP labelling in the adrenal gland from B1216 (original magnification ×20). B: Abnormal PrP in the secondary follicles of the prescapular lymph node from B1217 (original magnification ×10). C: Widespread distribution of Abnormal PrP in the obex from B1217 (original magnification ×10). D: Very localised deposition of Abnormal PrP in the spindles of the semitendinous muscle from B1216 (original magnification ×40). E and F: examples of different level of deposition of PrP^d in the renal papillae from respectively B1223 and B1217 (original magnification E ×20 and F×40).</p

Converting activity in the inocula from 1216B sheep analysed by vPMCA.

<p>Western blot analysis of the amplified products was carried out after the 3^rd, 5^th, 7^th and 9^th rounds, as indicated. The results obtained with the brain dilution curve are reported in left panels, those from visceral tissue inocula are in right panels. Blots were probed with SAF84 primary antibody.</p

Relative intensity of chemiluminescent signal of PrPSc in tissues from 4 ARQ/ARQ sheep measured from Western blot image data (see adjacent panel) of edible tissues compared with a 12.5% dilution of brain sample by three western blot methods.

<p>(a) BioRad TeSeE Western Blot, (b) centrifugal concentration, (c) NaPTA precipitation. Red diamonds indicate high signal; yellow – low signal; blue – negative.</p

Summary of the IHC, WB, ELISA and bioassay results.

<p>IHC: Score denotes the abundance of PrP^d in each visceral tissue; no labelling (−), mild (+), moderate(++) or severe accumulation (+++) of PrP^d. For muscle samples a semi-quantitative scoring system was applied to no. of positively labelled spindles; 0 (−), 1 (+), 1–5 (++), >5 (+++)</p><p>WB: +1/ +2/ +3 denotes pos/neg results from three separate methods where 1 = BioRad; 2 = centrifugal concentration; 3 = NaPTA</p><p>E = ELISA: +1/ +2 denotes pos/neg results from two separate methods where 1 = BioRad; 2 = IDEXX</p><p>VB = Vole bioassay results were categorised as follows (+++) = similar to brain dilution-3 or more efficient,</p><p>(++) = between brain dilutions −3 and −4, (+) = similar to brain dilution −4 or less efficient</p><p>Summary of the IHC, WB, ELISA and bioassay results.</p