J Vet Sci.  2015 Jun;16(2):179-186. 10.4142/jvs.2015.16.2.179.

Classical natural ovine scrapie prions detected in practical volumes of blood by lamb and transgenic mouse bioassays

• Dassanayake RP
• Truscott T
• Zhuang D
• Schneider
• Madsen-Bouterse SA
• Young AJ
• Stanton JB
• Davis WC
• O'Rourke K

• KMID: 2164527
• DOI: http://doi.org/10.4142/jvs.2015.16.2.179

Abstract

Scrapie is diagnosed antemortem in sheep by detecting misfolded isoforms of prion protein (PrP(Sc)) in lymphoid follicles of the rectal mucosa and nictitating membranes. Assay sensitivity is limited if (a) the biopsy is collected early during disease development, (b) an insufficient number of follicles is collected, or (c) peripheral accumulation of PrP(Sc) is reduced or delayed. A blood test would be convenient for mass live animal scrapie testing. Currently approved techniques, however, have their own detection limits. Novel detection methods may soon offer a non-animal-based, rapid platform with detection sensitivities that rival the prion bioassay. In anticipation, we sought to determine if diseased animals could be routinely identified with a bioassay using B lymphocytes isolated from blood sample volumes commonly collected for diagnostic purposes in small ruminants. Scrapie transmission was detected in five of six recipient lambs intravenously transfused with B lymphocytes isolated from 5~10 mL of blood from a naturally scrapie-infected sheep. Additionally, scrapie transmission was observed in 18 ovinized transgenic Tg338 mice intracerebrally inoculated with B lymphocytes isolated from 5~10 mL of blood from two naturally scrapie-infected sheep. Based on our findings, we anticipate that these blood sample volumes should be of diagnostic value.

Keyword

B lymphocytes; blood; prions; scrapie; Tg338 mice

MeSH Terms

Animals
B-Lymphocytes/*pathology
Biological Assay/*veterinary
Mice
Mice, Transgenic
Prions/*blood
Scrapie/blood/*diagnosis/transmission
Sheep
Prions

Figure

• 

  Fig. 1 Detection of PrPSc-specific immunolabeling in the lymphoid tissues of inoculated sheep. PrPSc (dark red) was visible in the follicles of retropharyngeal lymph nodes collected during necropsy from sheep inoculated with PBMCs (A) or B lymphocytes recovered from 10 (B) and 5 mL (C) of blood. PrPSc immunolabeling was not detected in uninoculated control sheep (D). Immunohistochemistry (IHC) was performed using a mixture of mAbs F99/97.6.1. and F89/160.1.5. (2.5 µg/mL each) with 3-amino-9-ethylcarbazole (AEC) chromagen. Scale bar = 100 µm

• 

  Fig. 2 Representative Western immunoblots for detecting PrPres in Tg338 mice inoculated with PBMCs and B lymphocytes. (A) PrPres-specific bands were detected for the brain homogenates of preclinical scrapie donor sheep 4125 (lane 1) along with brain homogenates of Tg338 mice inoculated with PBMCs (lanes 2 and 3), CD72+ B lymphocytes (lanes 4 and 5), and CD21+ B lymphocytes (lanes 6 and 7). These bands were not observed for the uninoculated control mouse (lane 8). (B) PrPres bands were also detected for the brain homogenates of clinical scrapie donor sheep 4124 (lane 1) as well as spleen homogenates of Tg338 mice inoculated with sIgM+ B lymphocytes (lanes 2~4) and CD21+ B lymphocytes (lanes 5~7). These bands were not observed for the uninoculated control mouse (lane 8). mAb F99/97.6.1 (2.5 µg/mL) was used to detect PrPres.

Reference

1. Andréoletti O, Berthon P, Marc D, Sarradin P, Grosclaude J, van Keulen L, Schelcher F, Elsen JM, Lantier F. Early accumulation of PrP^Sc in gut-associated lymphoid and nervous tissues of susceptible sheep from a Romanov flock with natural scrapie. J Gen Virol. 2000; 81:3115–3126.

2. Andréoletti O, Litaise C, Simmons H, Corbiére F, Lugan S, Costes P, Schelcher F, Vilette D, Grassi J, Lacroux C. Highly efficient prion transmission by blood transfusion. PLoS Pathog. 2012; 8:e1002782.

3. Andréoletti O, Orge L, Benestad SL, Beringue V, Litaise C, Simon S, Le Dur A, Laude H, Simmons H, Lugan S, Corbiére F, Costes P, Morel N, Schelcher F, Lacroux C. Atypical/Nor98 scrapie infectivity in sheep peripheral tissues. PLoS Pathog. 2011; 7:e1001285.

4. Bannach O, Birkmann E, Reinartz E, Jaeger KE, Langeveld JP, Rohwer RG, Gregori L, Terry LA, Willbold D, Riesner D. Detection of prion protein particles in blood plasma of scrapie infected sheep. PLoS One. 2012; 7:e36620.

5. Bolton DC, McKinley MP, Prusiner SB. Identification of a protein that purifies with the scrapie prion. Science. 1982; 218:1309–1311.

6. Dassanayake RP, Schneider DA, Truscott TC, Young AJ, Zhuang D, O'Rourke KI. Classical scrapie prions in ovine blood are associated with B lymphocytes and platelet-rich plasma. BMC Vet Res. 2011; 7:75.

7. Dassanayake RP, Truscott TC, Ozyigit MO, Zhuang D, Schneider DA, O'Rourke KI. Accumulation profiles of PrP^Sc in hemal nodes of naturally and experimentally scrapie- infected sheep. BMC Vet Res. 2013; 9:82.

8. Dennis MM, Thomsen BV, Marshall KL, Hall SM, Wagner BA, Salman MD, Norden DK, Gaiser C, Sutton DL. Evaluation of immunohistochemical detection of prion protein in rectoanal mucosa-associated lymphoid tissue for diagnosis of scrapie in sheep. Am J Vet Res. 2009; 70:63–72.

9. Dobie K, Barron R. Dissociation between transmissible spongiform encephalopathy (TSE) infectivity and proteinase K-resistant PrP^Sc levels in peripheral tissue from a murine transgenic model of TSE disease. J Virol. 2013; 87:5895–5903.

10. Edwards JC, Moore SJ, Hawthorn JA, Neale MH, Terry LA. PrP^Sc is associated with B cells in the blood of scrapie-infected sheep. Virology. 2010; 405:110–119.

11. Elder AM, Henderson DM, Nalls AV, Wilham JM, Caughey BW, Hoover EA, Kincaid AE, Bartz JC, Mathiason CK. In vitro detection of prionemia in TSE-infected cervids and hamsters. PloS One. 2013; 8:e80203.

12. Espenes A, Press CM, Landsverk T, Tranulis MA, Aleksandersen M, Gunnes G, Benestad SL, Fuglestveit R, Ulvund MJ. Detection of PrP^Sc in rectal biopsy and necropsy samples from sheep with experimental scrapie. J Comp Pathol. 2006; 134:115–125.

13. González L, Dagleish MP, Martin S, Dexter G, Steele P, Finlayson J, Jeffrey M. Diagnosis of preclinical scrapie in live sheep by the immunohistochemical examination of rectal biopsies. Vet Rec. 2008; 162:397–403.

14. Halliez S, Jaumain E, Huor A, Douet JY, Lugan S, Cassard H, Lacroux C, Béringue V, Andréoletti O, Vilette D. White blood cell-based detection of asymptomatic scrapie infection by ex vivo assays. PLoS One. 2014; 9:e104287.

15. Houston F, McCutcheon S, Goldmann W, Chong A, Foster J, Sisó S, González L, Jeffrey M, Hunter N. Prion diseases are efficiently transmitted by blood transfusion in sheep. Blood. 2008; 112:4739–4745.

16. Huang H, Rendulich J, Stevenson D, O'Rourke K, Balachandran A. Evaluation of Western blotting methods using samples with or without sodium phosphotungstic acid precipitation for diagnosis of scrapie and chronic wasting disease. Can J Vet Res. 2005; 69:193–199.

17. Konold T, Moore SJ, Bellworthy SJ, Simmons HA. Evidence of scrapie transmission via milk. BMC Vet Res. 2008; 4:14.

18. Lacroux C, Vilette D, Fernández-Borges N, Litaise C, Lugan S, Morel N, Corbiére F, Simon S, Simmons H, Costes P, Weisbecker JL, Lantier I, Lantier F, Schelcher F, Grassi J, Castilla J, Andréoletti O. Prionemia and leukocyte-platelet-associated infectivity in sheep transmissible spongiform encephalopathy models. J Virol. 2012; 86:2056–2066.

19. Mok SW, Proia RL, Brinkmann V, Mabbott NA. B cell-specific S1PR1 deficiency blocks prion dissemination between secondary lymphoid organs. J Immunol. 2012; 188:5032–5040.

20. O'Rourke KI, Baszler TV, Besser TE, Miller JM, Cutlip RC, Wells GAH, Ryder SJ, Parish SM, Hamir AN, Cockett NE, Jenny A, Knowles DP. Preclinical diagnosis of scrapie by immunohistochemistry of third eyelid lymphoid tissue. J Clin Microbiol. 2000; 38:3254–3259.

21. O'Rourke KI, Baszler TV, Miller JM, Spraker TR, Sadler-Riggleman I, Knowles DP. Monoclonal antibody F89/160.1.5 defines a conserved epitope on the ruminant prion protein. J Clin Microbiol. 1998; 36:1750–1755.

22. O'Rourke KI, Baszler TV, Parish SM, Knowles DP. Preclinical detection of PrP^Sc in nictitating membrane lymphoid tissue of sheep. Vet Rec. 1998; 142:489–491.

23. O'Rourke KI, Schneider DA, Spraker TR, Dassanayake RP, Highland MA, Zhuang D, Truscott TC. Transmissibility of caprine scrapie in ovine transgenic mice. BMC Vet Res. 2012; 8:42.

24. O'Rourke KI, Zhuang D, Truscott TC, Yan H, Schneider DA. Sparse PrP^Sc accumulation in the placentas of goats with naturally acquired scrapie. BMC Vet Res. 2011; 7:7.

25. Prusiner SB. Novel proteinaceous infectious particles cause scrapie. Science. 1982; 216:136–144.

26. Race R, Jenny A, Sutton D. Scrapie infectivity and proteinase K-resistant prion protein in sheep placenta, brain, spleen, and lymph node: implications for transmission and antemortem diagnosis. J Infect Dis. 1998; 178:949–953.

27. Ryder S, Dexter G, Bellworthy S, Tongue S. Demonstration of lateral transmission of scrapie between sheep kept under natural conditions using lymphoid tissue biopsy. Res Vet Sci. 2004; 76:211–217.

28. Schreuder BE, van Keulen LJ, Vromans ME, Langeveld JP, Smits MA. Tonsillar biopsy and PrP^Sc detection in the preclinical diagnosis of scrapie. Vet Rec. 1998; 142:564–568.

29. Terry LA, Howells L, Hawthorn J, Edwards JC, Moore SJ, Bellworthy SJ, Simmons H, Lizano S, Estey L, Leathers V, Everest SJ. Detection of PrP^Sc in blood from sheep infected with the scrapie and bovine spongiform encephalopathy agents. J Virol. 2009; 83:12552–12558.

30. Thorne L, Terry LA. In vitro amplification of PrP^Sc derived from the brain and blood of sheep infected with scrapie. J Gen Virol. 2008; 89:3177–3184.

31. van Keulen LJ, Schreuder BE, Meloen RH, Mooij-Harkes G, Vromans ME, Langeveld JP. Immunohistochemical detection of prion protein in lymphoid tissues of sheep with natural scrapie. J Clin Microbiol. 1996; 34:1228–1231.

32. van Keulen LJ, Schreuder BE, Vromans ME, Langeveld JP, Smits MA. Pathogenesis of natural scrapie in sheep. Arch Virol Suppl. 2000; 16:57–71.

33. Vilotte JL, Soulier S, Essalmani R, Stinnakre MG, Vaiman D, Lepourry L, Da Silva JC, Besnard N, Dawson M, Buschmann A, Groschup M, Petit S, Madelaine MF, Rakatobe S, Le Dur A, Vilette D, Laude H. Markedly increased susceptibility to natural sheep scrapie of transgenic mice expressing ovine PrP. J Virol. 2001; 75:5977–5984.

34. Young AJ, Marston WL, Dudler L. Subset-specific regulation of the lymphatic exit of recirculating lymphocytes in vivo. J Immunol. 2000; 165:3168–3174.