Classical scrapie prions are associated with peripheral blood monocytes and T-lymphocytes from naturally infected sheep

Abstract

Background: Classical scrapie is a transmissible spongiform encephalopathy (TSE) that affects sheep and goats. Our previous bioassay studies in lambs revealed that scrapie prions could be detected in association with peripheral blood monocular cells (PBMC), B lymphocytes and platelet-rich plasma fractions. In the present study, bioassay in lambs was again used to determine if scrapie prions are associated with the other two subsets of PBMC, monocytes and T lymphocytes.

Results: PBMC, monocytes and T lymphocytes were isolated from two preclinically affected VRQ/VRQ sheep naturally infected with classical ovine scrapie and intravenously transfused into VRQ/VRQ lambs post-weaning. As determined using standard immunohistochemistry for scrapie, abnormal isoforms of prion protein were detected in lymphoid tissues of lambs inoculated with PBMC (4/4 recipient lambs), monocytes (2/5) and T lymphocytes (1/4). Prion protein misfolding activity was detected by serial protein misfolding cyclic amplification (sPMCA) in PBMC from monocyte and T lymphocyte recipient sheep in agreement with antemortem rectal biopsy results, but such prion protein misfolding activity was not detected from other recipients.

Conclusions: These findings show that scrapie prions are associated with monocytes and T lymphocytes circulating in the peripheral blood of sheep naturally infected with classical scrapie. Combined with our previous findings, we can now conclude that all three major subsets of PBMC can harbor prions during preclinical disease and thus, present logical targets for development of a sensitive assay to detect scrapie prions. In this regard, we have also demonstrated that sPMCA can be used to detect scrapie prions associated with PBMC.

Fig. 1

Detection of PrP^Sc immunolabeling in the follicles of rectoanal mucosa-associated lymphoid tissues of donor and recipient sheep. Note the PrP^Sc immunolabeling (dark red) was visible in the RAMALT follicles of donor sheep (a, animal ID: 4454) and recipient sheep transfused with PBMC (b, animal ID: 4601), monocytes (c, animal ID: 4595) or pan T lymphocytes (d, animal ID: 4590). IHC was performed using a mixture of prion mAbs F99/97.6.1 and F89/160.1.5. (2.5 μg/mL each) and AEC chromogen

Fig. 2

Prion protein misfolding activity detected by sPMCA in PBMC of recipient sheep transfused with monocytes or T lymphocytes from scrapie-affected sheep. PBMC (1x10⁷ cells) were added to mNBH and subjected to six PMCA rounds of 48 cycles of sonication and incubation. Samples were diluted 1:3 into fresh mNBH between rounds. Pre- and post-PMCA samples underwent proteinase K digestion (200 μg/ml for 90 min at 37 °C) prior to western blot analysis with prion mAb P4. Representative western blots are shown. a PBMC prepared from positive control scrapie sheep (4124) pre- and post-sPMCA (lanes 1 and 2, respectively). PBMC prepared from monocyte recipient sheep (lane 4 = 4546, lane 5 = 4593, lane 6 = 4594, lane 7 = 4595, lane 9 = 4597) and an uninoculated control sheep (lane 8 = 4596). A scrapie naïve sheep (lane 3 = 4545) and mNBH (lane = 10) served as negative controls for sPMCA. b PBMC from positive control scrapie sheep (4124) is shown pre- and post-sPMCA (lanes 1 and 2, respectively). PBMC prepared from T lymphocyte recipient sheep (lane 4 = 4539, lane 5 = 4588, lane 6 = 4589, lane 7 = 4590) and uninoculated control sheep (lane 8 = 4591, lane 9 = 4597); and mNBH (negative sPMCA control, lane = 3). Molecular mass markers (in kDa) are indicated on the left of the blots