Removal of exogenous and endogenous prion infectivity from red blood cell preparations of scrapie-infected hamster blood by leukoreduction filters resulted in significant reduction of scrapie infection in hamsters following transfusion . Leukodepletion significantly reduced the risk of vCJD transmission in human following blood transfusion recipients as well [17, 18]. However, a recent study by McCutcheon et al., (2011) revealed that leucoreduction did not prevent the BSE transmission to sheep following a single blood transfusion . Detection of PrPSc labeling in the lymphoid tissues and development of clinical scrapie in whole blood and buffy coat transfusion recipients in this study as well as in previous studies [8–10] confirmed that prion infectivity is associated with blood from classical scrapie infected sheep. Although previous sheep blood transfusion studies used a relatively large volume of whole blood (400 - 500 mL), volumes of 50 - 135 mL whole blood from scrapie infected sheep were sufficient to transmit scrapie infection to the most recipients in this study. It is difficult to avoid the loss of PBMCs during density-gradient cell separation and MACS-based cell enrichment procedures (CD72+ and/or CD21+ B lymphocytes) due to the multiple washing steps involved and the inherent limitation to the cell-binding capacity of the columns. Therefore, it is possible that even much lower volumes of scrapie infected sheep blood might be sufficient to cause infection in sheep. Demonstration of prion infectivity in much smaller volumes of blood may be helpful in development of conventional blood-based diagnostic testing for scrapie in sheep.
We used standard IHC detection of PrPSc in tissues as a surrogate marker for transmission of infectivity to preclinical recipient sheep. Analysis of antemortem and postmortem lymphoid tissues in sheep receiving transfusion with the PBMC fractions were confirmed positive for PrPSc immunolabeling indicating that prion infectivity is associated with PBMC fraction of sheep blood. Previous studies showed PrPSc was detected from PBMCs [12, 13] and a subpopulation of B lymphocytes . CD72 has been identified as a pan B lymphocyte marker in sheep  and also in mice . Approximately 50% of adult sheep B lymphocytes are positive for CD21 . A significant proportion of peripheral lymphocytes recirculate continuously between the blood, the lymph and the tissues. The lymph nodes are the major site of exchange for recirculating lymphocytes between the blood and the lymph. The migration competent or recirculating B lymphocytes readily migrate into the lymphatic recirculation pathway due to the cell surface expression of CD21 and CD62L or L-selectin . Therefore, we used anti-CD72 and anti-CD21 mAbs to isolate pan B lymphocytes and recirculating B lymphocytes from the PBMCs fraction, respectively. Transfusion of CD72+ B lymphocytes or CD21+ B lymphocytes from scrapie infected sheep resulted in PrPSc detection in lymphoid tissues of recipients. These results are consistent with PrPSc being detected in MHC class II DQ+, sIgM+, CD11b+, CD11c+ and CD21+/- B lymphocytes in sheep , and CD72+ B lymphocytes harboring infectious CWD prions in white-tailed deer .
The present study utilized a short observation period in the natural host as a model biased for quick detection of blood fractions having relatively high prion levels. The platelet-poor plasma fraction from scrapie infected sheep did not contain adequate levels of prion infectivity for detection in this model. Two of the three MARQ/MARQ lambs transfused with platelet rich plasma were positive by rectal biopsy 218 and 288 post-transfusion, approximately the same interval seen in MARQ/MARQ lambs transfused with whole blood (222-252 dpt, n = 4) or buffy coat cells (239-288 days, n = 2). CWD and BSE infectivity were associated with platelets or platelet-rich plasma pellets in white-tailed deer  and sheep , respectively. However, hamsters inoculated with platelets from scrapie infected hamsters did not develop scrapie . The possibility exists that rodent models and ruminants have slightly different cells that contain infectivity or the processing of platelets could affect scrapie infectivity. Other studies have suggested that there is cell-free PrPSc in sheep plasma [15, 22]. Passage of radioactively labeled, highly purified murine PrPSc through the mouse blood-brain barrier  and detection of PrPSc in sheep circumventricular organs that lack a blood-brain barrier  suggest the possibility of cell-free PrPSc in the blood. Detection of PrPSc by PMCA in hamster plasma samples devoid of platelets suggests the possibility of cell-free PrPSc in hamster plasma [25, 26]. Similarly, vCJD infectivity in plasma components has been reported from human donors suggesting cell-free plasma can also carry infectious vCJD prions [27, 28]. Given the similarity in pathogenesis between BSE and scrapie infection in sheep, one would expect similar outcome from both forms. However, a recent study by McCutcheon et al., (2011) with BSE-infected sheep revealed that platelet-poor plasma supernatant can also efficiently transmit prion infectivity to recipient lambs following a single blood transfusion . The detection of preclinical scrapie in recipient lambs took 594 to 1089 dpt. The lack of PrPSc detection in lymphoid tissues of platelet-poor plasma recipients in our study could have been due to a lower prion titer in platelet-poor plasma samples and/or necropsy of animals at 550 dpt. Poisson distribution of particles in solution indicates a far greater number of recipient animals per group would have been necessary to distinguish sample fraction "titers". In contrast to sheep, hamster and human findings, recent studies in white-tailed deer and cervidized transgenic mouse revealed that CWD infectivity was not associated with platelet-poor plasma, but with platelets . Although the presence of PrPSc in hamsters' plasma fraction is confirmed, prion infectivity in that fraction has not yet been reported.
Our previous study demonstrated that one nonsynonymous allele (T112) was associated with prolonged survival in MARQ/TARQ scrapie-exposed sheep compared to MARQ/MARQ sheep . Although the focus of this study was to identify which blood components carry prion infectivity, four MARQ/TARQ and four TARQ/MVRQ animals were included to assess whether T112 polymorphism might delay PrPSc detection in RAMALT follicles following infection by transfusion. Lack of PrPSc labeling in RAMALT follicles of six of eight recipients confirms that T112 polymorphism delayed prion accumulation in sheep rectal lymphoid tissues. Although the number of animals used in this study was limited, the delay in PrPSc accumulation in RAMALT particularly in MARQ/TARQ animals needs to be considered when scrapie diagnosis is determined.