Prionemia and leukocyte-platelet-associated infectivity in sheep transmissible spongiform encephalopathy models

Abstract

The dynamics of the circulation and distribution of transmissible spongiform encephalopathy (TSE) agents in the blood of infected individuals remain largely unknown. This clearly limits the understanding of the role of blood in TSE pathogenesis and the development of a reliable TSE blood detection assay. Using two distinct sheep scrapie models and blood transfusion, this work demonstrates the occurrence of a very early and persistent prionemia. This ability to transmit disease by blood transfusion was correlated with the presence of infectivity in white blood cells (WBC) and peripheral blood mononucleated cells (PBMC) as detected by bioassay in mice overexpressing the ovine prion protein PrP (tg338 mice) and with the identification of abnormal PrP in WBC after using protein misfolding cyclic amplification (PMCA). Platelets and a large variety of leukocyte subpopulations also were shown to be infectious. The use of endpoint titration in tg338 mice indicated that the infectivity in WBC (per ml of blood) was 10(6.5)-fold lower than that in 1 g of posterior brainstem sample. In both WBC and brainstem, infectivity displayed similar resistance to PK digestion. The data strongly support the concept that WBC are an accurate target for reliable TSE detection by PMCA. The presence of infectivity in short-life-span blood cellular elements raises the question of the origin of prionemia.

Fig 1

Lesion profiles in tg338 mice inoculated with brain material and blood fractions from sheep. Shown are lesion profiles (showing vacuolar changes) in tg338 mice inoculated by the intracerebral route with (A) 10% obex homogenate prepared from clinically affected VRQ/VRQ sheep that were naturally exposed to Langlade classical scrapie agent (▴; blood donor 2 in Table 1) or that were transfused with blood collected from donor 2 at 90 days (○) and 360 days (▿) of age; (B) 10% obex homogenate from clinically affected VRQ/VRQ sheep that were orally challenged with PG127 classical scrapie agent (△; blood donor 5 in Table 2) or that were transfused with blood collected from donor 4 at 90 days (○) and 190 days (▿) postinoculation; (C) white blood cell homogenates (white symbols) and PBMC (black symbols) prepared from VRQ/VRQ sheep that were naturally exposed to Langlade classical scrapie agent (blood donor 2 in Table 3) collected at 210 days (▿) and 360 days (○) of age; and (D) white blood cell homogenates (white symbols) and PBMC (black symbols) prepared from VRQ/VRQ sheep that were orally exposed to PG127 classical scrapie agent (blood donor 5 in Table 4) and collected 130 days (▿) and 180 days (○) postinoculation.

Fig 2

PrP^res Western blot of PG127 infected sheep brain and white blood cells amplified by PMCA. (A and B) Dilution series (1/10) in PBS of 10% brain homogenates prepared from a PG127 scrapie-affected sheep (using the same material as that used for titrations in Table 5) and two scrapie-negative VRQ/VRQ sheep were submitted to two successive PMCA rounds (rounds 1 [A] and 2 [B]). Tubes seeded with different dilutions were processed for PrP^res Western blotting detection (Sha31 anti-PrP antibody). Lane 1, Western blotting-positive control; lanes 2 to 8, 1/10 dilution series of PG127-positive brain homogenate (from 10⁻³ to 10⁻⁹); lanes 9 and 10, VRQ/VRQ TSE-free sheep (10⁻¹ dilution). (C to E) White blood cells from one VRQ/VRQ sheep (C and D, lanes 2 to 6) (donor 4 in Tables 2, 3, and 5) or one ARR/ARR sheep (E, lanes 2 to 6) (control 1 in Tables 2 and 4) that had been orally challenged with PG127 scrapie were prepared at different time points of the incubation period. WBC from two TSE-free VRQ/VRQ (C and D, lanes 7 and 8) and two TSE-free ARR/ARR sheep (E, lanes 7 and 8) were used as controls. All WBC samples were submitted to two successive PMCA rounds (rounds 1 [C] and 2 [D and E]), and amplicons then were processed for PrP^Sc Western blotting detection (Sha31 anti-PrP antibody). Lane 1, positive Western blotting control. Lanes 2 to 6 correspond to WBC prepared at 30, 60, 90, 130, and 190 days postinoculation, respectively, in PG127-challenged animals. Lanes 9 and 10 correspond to WBC prepared from TSE-free controls.