Pathological Prion Protein in the Tongues of Sheep
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病菌学杂志 2005年第9期
CEA, Italian Reference Laboratory for TSE, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Turin, Italy
ABSTRACT
Tongue involvement by prion spreading was shown to be a common outcome after oral or intracranial experimental challenge with scrapie and transmissible mink encephalopathy sources in rodent models. It is also known that bovine spongiform encephalopathy, which is pathogenic for humans, is experimentally transmissible to sheep and can lead to a disease indistinguishable from scrapie. A recent European Food Safety Authority opinion recommended research into PrPsc accumulation in the tongues of ruminants. We report on the detection of PrPsc in the tongues of seven scrapie-infected sheep by immunohistochemistry and Western blotting.
TEXT
Recent studies on rodent models (1, 8, 10), have reported the detection of PrPsc in tongue tissue after oral or intracranial experimental challenge with scrapie and transmissible mink encephalopathy sources. Unlike bovine spongiform encephalopathy, scrapie is not considered a risk to humans health (2), but it has been demonstrated that under experimental conditions sheep are easily infected by the bovine spongiform encephalopathy agent and that they carry abundant amounts of infectivity throughout most body tissues (6). A European Food Safety Authority opinion (3) recommends testing for PrPsc presence and accumulation in ruminant tongues in order to facilitate risk quantification and assessment. In this study, we report on the presence of PrPsc in the tongues of sheep infected with naturally occurring scrapie.
We studied 10 negative, regularly slaughtered adult sheep and 10 adult sheep positive by rapid test (Prionics-Check Western) coming from two different affected flocks in Piedmont and Tuscany, respectively (flock A, eight sheep of the Biellese breed, aged 20 months to 7 years; flock B, two sheep of the Sarda breed, aged 20 months and 2.5 years). The disease was confirmed by histology, immunohistochemistry (IHC), and Western blotting (WB) in the brainstem. Both sheep from flock B and four from flock A showed clinical signs of scrapie (tremors, emaciation, falling). No relevant differences in the amount of PrPsc in the obex were found between animals with or without clinical signs. All animals had an ARQ/ARQ PrP genotype.
The tongue of each animal was cut into two halves, of which one was fixed in buffered formalin and the other was frozen. Two specular areas of each half were examined independently: one at the level of the apex and one from the corpus linguae. For IHC, 10 serial tissue sections, 5 to 6 μm thick, were obtained and numbered: sections numbered 1, 4, and 7 were stained to detect PrPsc immunoreactivity; sections numbered 2, 5, and 8 and 3, 6, and 9, respectively, were stained using PGP 9.5 and neurofilament to investigate the possible involvement of cells and nerve fibers. All tissue sections were dewaxed and rehydrated by routine methods and then subjected to an antigen retrieval procedure (5). The sections were immersed in 98% formic acid for 20 min, washed in distilled water, and then autoclaved for 20 min at 121°C in distilled water. Endogenous peroxidase activity was blocked in 3% hydrogen peroxide for 20 min at room temperature. To remove nonspecific tissue antigens, the sections were incubated with 5% blocking serum for 20 min at room temperature. Tissues were incubated overnight at 4°C with the primary antibody. PrP immunostaining was carried out with monoclonal antibody (MAb) F99/97.6.1 (9) (1:1,000 dilution; VMRD Inc., Pullman, WA) or MAb L42 (4) (1:250 dilution; R-Biopharm, Darmstadt, Germany). Anti-PGP 9.5 polyclonal antibody (1:100 dilution; Dakocytomation, Carpinteria, CA) and anti-human triple neurofilament MAb (1:100 dilution; Dakocytomation, Carpinteria, CA) were used for nerve fiber identification. The rest of the immunohistochemical procedure was carried out using a commercial immunoperoxidase technique (Vectastain ABC kit; Vector, Burlingame, CA), using 3,3'-diaminobenzidine (Dakocytomation, Carpinteria, CA) as chromogen; the sections were then counterstained with Meyer's hematoxylin.
The epithelium with underlying connective tissue and the muscle layer were separated from the frozen material and examined separately by a highly sensitive WB using sodium phosphotungstic acid precipitation (11). Briefly, 10% (wt/vol) homogenates from 0.2 to 0.4 g of frozen brainstem and tongue were precipitated with sodium phosphotungstic acid (Sigma) and then digested with proteinase K (50 μg/ml) at 37°C for 30 min. After denaturation, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% acrylamide) and electroblotted onto polyvinylidene difluoride membrane. The blots were incubated with MAb P4 (7) (1:5,000 dilution; R-Biopharm, Darmstadt, Germany). Immunoreaction was revealed with chemiluminescent substrate (Roche; 25 mM).
In flock A, PrPsc was detected by WB and IHC in both areas of the tongues of five out of eight animals. Only one sheep showed clinical signs. No PrPsc was detected in the tongues of the remaining three sheep, although they were at the clinical stage of the disease. In flock B, both animals contained PrPsc in the tongue. (Table 1).
No immunoreaction was observed in the tongues of the 10 negative controls by either IHC or WB. PrP immunoreactivity was most prominent in the connective tissue core of the circumvallate and fungiform papillae, and it was occasionally observed in the basal region of the taste buds. PrP immunostaining showed a linear pattern corresponding to localization at the level of nerve processes, as revealed by anti-PGP 9.5 and anti-neurofilament immunostaining (Fig. 1).
PrPsc was detected by WB in both the epithelium and the muscle layers of six out of seven cases. In one animal, the epithelium samples were positive but the muscle samples tested negative (Fig. 2). To estimate the relative concentration of PrPsc in the tongues, we compared the intensities of the proteinase K-digested Western blot signals with calibration curves obtained by diluting the brainstems of the scrapie-positive sheep with a scrapie-negative tongue homogenate. The signal intensity of the positive tongues was generally similar to that obtained when 0.25 to 0.025 μg of a positive brainstem homogenate was diluted with 50 mg of a negative tongue homogenate. From this, we estimated that the levels of PrPsc in the examined tongues were lower than those found in the corresponding brainstem by a factor of approximately 2 x 10–5 to 2 x 10–6 (Fig. 3).
To our knowledge, this is the first report of PrPsc deposition detected in the lingual papillae of the tongues of sheep infected with naturally occurring scrapie. Further investigations are required to establish the onset of tongue involvement in the course of the disease. Although the rate of tongue involvement cannot be assessed yet, our findings suggest a need for revising the distribution of scrapie infectivity in sheep in peripheral tissues.
ACKNOWLEDGMENTS
We thank Massimo Tabaton, Gianluigi Zanusso, and Marion Simmons for critical reading of the manuscript.
This work was funded by Italian Ministry of Health grant IZSPLV004/01.
REFERENCES
Bartz, J. C., A. E. Kincaid, and R. A. Bessen. 2003. Rapid prion neuroinvasion following tongue infection. J. Virol. 77:583-591.
Brown, P., and R. Bradley. 1998. 1755 and all that: a historical primer of transmissible spongiform encephalopathy. BMJ 317:1688-1692.
European Food Safety Authority. 2004. Opinion of the Scientific Panel on Biological Hazards of the European Food Safety Authority on BSE risk from bovine tonsil and consumption of bovine tongue. EFSA J. 41:1-4.
Hardt, M., T. Baron, and M. H. Groschup. 2000. A comparative study of immunohistochemical methods for detecting abnormal prion protein with monoclonal and polyclonal antibodies. J. Comp. Pathol. 122:43-53.
Haritani, M., Y. I. Spencer, and G. A. H. Wells. 1994. Hydrated pretreatment enhancement of prion protein immunoreactivity in formalin fixed bovine spongiform encephalopathy affected brain. Acta Neuropathol. 87:86-90.
Jeffrey, M., S. Ryder, S. Martin, S. A. C. Hawkins, L. Terry, C. Berthelin-Baker, and S. J. Bellworthy. 2001. Oral inoculation of sheep with the agent of bovine spongiform encephalopathy (BSE). 1. Onset and distribution of disease-specific PrP accumulation in brain and viscera. J. Comp. Pathol. 124:280-289.
Madec, J., M. H. Groschup, A. Buschmann, P. Belli, D. Cavalas, and T. Baron. 1998. Sensitivity of the Western blot detection of prion protein PrPres in natural sheep scrapie. J. Virol. Methods 75:169-177.
Mulcahy, E. R., J. C. Bartz, A. E. Kincaid, and R. A. Bessen. 2004. Prion infection of skeletal muscle cells and papillae in the tongue. J. Virol. 78:6792-6798.
Spraker, T. R., K. I. O'Rourke, A. Balachandran, R. R. Zink, B. A. Cummings, M. W. Miller, and B. E. Powers. 2002. Validation of monoclonal antibody F99/97.6.1 for immunohistochemical staining of brain and tonsil in mule deer (Odocoileus hemionus) with chronic wasting disease. J. Vet. Diagn. Investig. 14:3-7.
Thomzig, A., C. Kratzel, G. Lenz, D. Kruger, and M. Beekes. 2003. Widespread PrPsc accumulation in muscles of hamsters orally infected with scrapie. EMBO Rep. 4:530-533.
Wadsworth, J. D. F., S. Joiner, A. F. Hill, T. A. Campbell, M. Desbruslais, P. J. Luthert, and J. Collinge. 2001. Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay. Lancet 358:171-180.(Cristina Casalone, Cristi)
ABSTRACT
Tongue involvement by prion spreading was shown to be a common outcome after oral or intracranial experimental challenge with scrapie and transmissible mink encephalopathy sources in rodent models. It is also known that bovine spongiform encephalopathy, which is pathogenic for humans, is experimentally transmissible to sheep and can lead to a disease indistinguishable from scrapie. A recent European Food Safety Authority opinion recommended research into PrPsc accumulation in the tongues of ruminants. We report on the detection of PrPsc in the tongues of seven scrapie-infected sheep by immunohistochemistry and Western blotting.
TEXT
Recent studies on rodent models (1, 8, 10), have reported the detection of PrPsc in tongue tissue after oral or intracranial experimental challenge with scrapie and transmissible mink encephalopathy sources. Unlike bovine spongiform encephalopathy, scrapie is not considered a risk to humans health (2), but it has been demonstrated that under experimental conditions sheep are easily infected by the bovine spongiform encephalopathy agent and that they carry abundant amounts of infectivity throughout most body tissues (6). A European Food Safety Authority opinion (3) recommends testing for PrPsc presence and accumulation in ruminant tongues in order to facilitate risk quantification and assessment. In this study, we report on the presence of PrPsc in the tongues of sheep infected with naturally occurring scrapie.
We studied 10 negative, regularly slaughtered adult sheep and 10 adult sheep positive by rapid test (Prionics-Check Western) coming from two different affected flocks in Piedmont and Tuscany, respectively (flock A, eight sheep of the Biellese breed, aged 20 months to 7 years; flock B, two sheep of the Sarda breed, aged 20 months and 2.5 years). The disease was confirmed by histology, immunohistochemistry (IHC), and Western blotting (WB) in the brainstem. Both sheep from flock B and four from flock A showed clinical signs of scrapie (tremors, emaciation, falling). No relevant differences in the amount of PrPsc in the obex were found between animals with or without clinical signs. All animals had an ARQ/ARQ PrP genotype.
The tongue of each animal was cut into two halves, of which one was fixed in buffered formalin and the other was frozen. Two specular areas of each half were examined independently: one at the level of the apex and one from the corpus linguae. For IHC, 10 serial tissue sections, 5 to 6 μm thick, were obtained and numbered: sections numbered 1, 4, and 7 were stained to detect PrPsc immunoreactivity; sections numbered 2, 5, and 8 and 3, 6, and 9, respectively, were stained using PGP 9.5 and neurofilament to investigate the possible involvement of cells and nerve fibers. All tissue sections were dewaxed and rehydrated by routine methods and then subjected to an antigen retrieval procedure (5). The sections were immersed in 98% formic acid for 20 min, washed in distilled water, and then autoclaved for 20 min at 121°C in distilled water. Endogenous peroxidase activity was blocked in 3% hydrogen peroxide for 20 min at room temperature. To remove nonspecific tissue antigens, the sections were incubated with 5% blocking serum for 20 min at room temperature. Tissues were incubated overnight at 4°C with the primary antibody. PrP immunostaining was carried out with monoclonal antibody (MAb) F99/97.6.1 (9) (1:1,000 dilution; VMRD Inc., Pullman, WA) or MAb L42 (4) (1:250 dilution; R-Biopharm, Darmstadt, Germany). Anti-PGP 9.5 polyclonal antibody (1:100 dilution; Dakocytomation, Carpinteria, CA) and anti-human triple neurofilament MAb (1:100 dilution; Dakocytomation, Carpinteria, CA) were used for nerve fiber identification. The rest of the immunohistochemical procedure was carried out using a commercial immunoperoxidase technique (Vectastain ABC kit; Vector, Burlingame, CA), using 3,3'-diaminobenzidine (Dakocytomation, Carpinteria, CA) as chromogen; the sections were then counterstained with Meyer's hematoxylin.
The epithelium with underlying connective tissue and the muscle layer were separated from the frozen material and examined separately by a highly sensitive WB using sodium phosphotungstic acid precipitation (11). Briefly, 10% (wt/vol) homogenates from 0.2 to 0.4 g of frozen brainstem and tongue were precipitated with sodium phosphotungstic acid (Sigma) and then digested with proteinase K (50 μg/ml) at 37°C for 30 min. After denaturation, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% acrylamide) and electroblotted onto polyvinylidene difluoride membrane. The blots were incubated with MAb P4 (7) (1:5,000 dilution; R-Biopharm, Darmstadt, Germany). Immunoreaction was revealed with chemiluminescent substrate (Roche; 25 mM).
In flock A, PrPsc was detected by WB and IHC in both areas of the tongues of five out of eight animals. Only one sheep showed clinical signs. No PrPsc was detected in the tongues of the remaining three sheep, although they were at the clinical stage of the disease. In flock B, both animals contained PrPsc in the tongue. (Table 1).
No immunoreaction was observed in the tongues of the 10 negative controls by either IHC or WB. PrP immunoreactivity was most prominent in the connective tissue core of the circumvallate and fungiform papillae, and it was occasionally observed in the basal region of the taste buds. PrP immunostaining showed a linear pattern corresponding to localization at the level of nerve processes, as revealed by anti-PGP 9.5 and anti-neurofilament immunostaining (Fig. 1).
PrPsc was detected by WB in both the epithelium and the muscle layers of six out of seven cases. In one animal, the epithelium samples were positive but the muscle samples tested negative (Fig. 2). To estimate the relative concentration of PrPsc in the tongues, we compared the intensities of the proteinase K-digested Western blot signals with calibration curves obtained by diluting the brainstems of the scrapie-positive sheep with a scrapie-negative tongue homogenate. The signal intensity of the positive tongues was generally similar to that obtained when 0.25 to 0.025 μg of a positive brainstem homogenate was diluted with 50 mg of a negative tongue homogenate. From this, we estimated that the levels of PrPsc in the examined tongues were lower than those found in the corresponding brainstem by a factor of approximately 2 x 10–5 to 2 x 10–6 (Fig. 3).
To our knowledge, this is the first report of PrPsc deposition detected in the lingual papillae of the tongues of sheep infected with naturally occurring scrapie. Further investigations are required to establish the onset of tongue involvement in the course of the disease. Although the rate of tongue involvement cannot be assessed yet, our findings suggest a need for revising the distribution of scrapie infectivity in sheep in peripheral tissues.
ACKNOWLEDGMENTS
We thank Massimo Tabaton, Gianluigi Zanusso, and Marion Simmons for critical reading of the manuscript.
This work was funded by Italian Ministry of Health grant IZSPLV004/01.
REFERENCES
Bartz, J. C., A. E. Kincaid, and R. A. Bessen. 2003. Rapid prion neuroinvasion following tongue infection. J. Virol. 77:583-591.
Brown, P., and R. Bradley. 1998. 1755 and all that: a historical primer of transmissible spongiform encephalopathy. BMJ 317:1688-1692.
European Food Safety Authority. 2004. Opinion of the Scientific Panel on Biological Hazards of the European Food Safety Authority on BSE risk from bovine tonsil and consumption of bovine tongue. EFSA J. 41:1-4.
Hardt, M., T. Baron, and M. H. Groschup. 2000. A comparative study of immunohistochemical methods for detecting abnormal prion protein with monoclonal and polyclonal antibodies. J. Comp. Pathol. 122:43-53.
Haritani, M., Y. I. Spencer, and G. A. H. Wells. 1994. Hydrated pretreatment enhancement of prion protein immunoreactivity in formalin fixed bovine spongiform encephalopathy affected brain. Acta Neuropathol. 87:86-90.
Jeffrey, M., S. Ryder, S. Martin, S. A. C. Hawkins, L. Terry, C. Berthelin-Baker, and S. J. Bellworthy. 2001. Oral inoculation of sheep with the agent of bovine spongiform encephalopathy (BSE). 1. Onset and distribution of disease-specific PrP accumulation in brain and viscera. J. Comp. Pathol. 124:280-289.
Madec, J., M. H. Groschup, A. Buschmann, P. Belli, D. Cavalas, and T. Baron. 1998. Sensitivity of the Western blot detection of prion protein PrPres in natural sheep scrapie. J. Virol. Methods 75:169-177.
Mulcahy, E. R., J. C. Bartz, A. E. Kincaid, and R. A. Bessen. 2004. Prion infection of skeletal muscle cells and papillae in the tongue. J. Virol. 78:6792-6798.
Spraker, T. R., K. I. O'Rourke, A. Balachandran, R. R. Zink, B. A. Cummings, M. W. Miller, and B. E. Powers. 2002. Validation of monoclonal antibody F99/97.6.1 for immunohistochemical staining of brain and tonsil in mule deer (Odocoileus hemionus) with chronic wasting disease. J. Vet. Diagn. Investig. 14:3-7.
Thomzig, A., C. Kratzel, G. Lenz, D. Kruger, and M. Beekes. 2003. Widespread PrPsc accumulation in muscles of hamsters orally infected with scrapie. EMBO Rep. 4:530-533.
Wadsworth, J. D. F., S. Joiner, A. F. Hill, T. A. Campbell, M. Desbruslais, P. J. Luthert, and J. Collinge. 2001. Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay. Lancet 358:171-180.(Cristina Casalone, Cristi)