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Biochemical typing of pathological prion protein in aging cattle with BSE.

Tester S, Juillerat V, Doherr MG, Haase B, Polak M, Ehrensperger F, Leeb T, Zurbriggen A, Seuberlich T - Virol. J. (2009)

Bottom Line: Our work confirmed H-type BSE in a zebu but classified all other cases as C-type BSE; indicating a very low incidence of H- and L-type BSE in Switzerland.It was documented for the first time that the biochemical PrPres type was consistent across different brain regions of aging animals with C-type and H-type BSE, i.e. independent of the neuroanatomical structure investigated.Taken together this study provides further characteristics of the BSE epidemic in Switzerland and generates new baseline data for the definition of C- and H-type BSE phenotypes, thereby underpinning the notion that they indeed represent distinct prion disease entities.

View Article: PubMed Central - HTML - PubMed

Affiliation: NeuroCenter, Reference Laboratory for TSE in animals, Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Berne, Switzerland. seraina.tester@itn.unibe.ch

ABSTRACT

Background: The broad enforcement of active surveillance for bovine spongiform encephalopathy (BSE) in 2000 led to the discovery of previously unnoticed, atypical BSE phenotypes in aged cattle that differed from classical BSE (C-type) in biochemical properties of the pathological prion protein. Depending on the molecular mass and the degree of glycosylation of its proteinase K resistant core fragment (PrPres), mainly determined in samples derived from the medulla oblongata, these atypical cases are currently classified into low (L)-type or high (H)-type BSE. In the present study we address the question to what extent such atypical BSE cases are part of the BSE epidemic in Switzerland.

Results: To this end we analyzed the biochemical PrPres type by Western blot in a total of 33 BSE cases in cattle with a minimum age of eight years, targeting up to ten different brain regions. Our work confirmed H-type BSE in a zebu but classified all other cases as C-type BSE; indicating a very low incidence of H- and L-type BSE in Switzerland. It was documented for the first time that the biochemical PrPres type was consistent across different brain regions of aging animals with C-type and H-type BSE, i.e. independent of the neuroanatomical structure investigated.

Conclusion: Taken together this study provides further characteristics of the BSE epidemic in Switzerland and generates new baseline data for the definition of C- and H-type BSE phenotypes, thereby underpinning the notion that they indeed represent distinct prion disease entities.

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Biochemical PrPres typing in diagnostic target sites of aged cattle with BSE. PrPres was analyzed at least five times on different gels with a core-binding antibody (mAb 6H4) by Western blot. a) Average molecular masses of the unglycosylated (red open circles), the monoglycosylated (green filled circles) and the diglycosylated band (blue rectangles) of PrPres are depicted with the related standard errors of the mean (S.E.M.). The cut-off value for the molecular mass of the unglycosylated band to discriminate H-type from C-type BSE was defined as the average molecular mass of all samples under investigation (solid line) +/- 5% (dashed line). b) Tri-plot graph presenting the relative intensities of the un-, mono- and diglycosylated PrPres moieties. The L-type BSE control (green square) is the sole sample that fell below the decision limit of 55% relative intensity of the diglycosylated PrPres (blue dashed line). The H-type BSE control is indicated by a red triangle and the zebu Charly-04 by a yellow circle. Samples were derived from medulla oblongata at the level of obex but where this was not available hippocampus was examined (marked by asterisks). CS, clinical suspect; ES, emergency slaughter; FS, fallen stock; RS, routine slaughter.
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Figure 2: Biochemical PrPres typing in diagnostic target sites of aged cattle with BSE. PrPres was analyzed at least five times on different gels with a core-binding antibody (mAb 6H4) by Western blot. a) Average molecular masses of the unglycosylated (red open circles), the monoglycosylated (green filled circles) and the diglycosylated band (blue rectangles) of PrPres are depicted with the related standard errors of the mean (S.E.M.). The cut-off value for the molecular mass of the unglycosylated band to discriminate H-type from C-type BSE was defined as the average molecular mass of all samples under investigation (solid line) +/- 5% (dashed line). b) Tri-plot graph presenting the relative intensities of the un-, mono- and diglycosylated PrPres moieties. The L-type BSE control (green square) is the sole sample that fell below the decision limit of 55% relative intensity of the diglycosylated PrPres (blue dashed line). The H-type BSE control is indicated by a red triangle and the zebu Charly-04 by a yellow circle. Samples were derived from medulla oblongata at the level of obex but where this was not available hippocampus was examined (marked by asterisks). CS, clinical suspect; ES, emergency slaughter; FS, fallen stock; RS, routine slaughter.

Mentions: To investigate the PrPres phenotype in detail we initially analyzed samples from the medulla oblongata (or from the hippocampus when MO was not available) in WB protocol I and we determined the molecular masses and relative proportions of the PrPres glycoforms. Only the H-type control and the H-type zebu revealed an unglycosylated PrPres band of an average molecular mass above 19 kDa with mAb 6H4. All other animals including the L-type control showed an average molecular mass in the range of 17.5 kDa to 18.82 kDa, which lies within the decision limit of ± 5% of the overall average molecular mass (Figure 2a). The average relative intensity of the diglycosylated band in all aging cattle with BSE was above the decision limit of 55% (57.5% to 64.9%) and for the L-type, as expected, much lower at 49.9% (Figure 2b). None of the cattle samples showed considerable reactivity with the N-terminal mAb 12B2 and all displayed a three-band pattern with mAb SAF84 (data not shown). Hence, the PrPres phenotype of the remaining 32 Swiss cattle included in this study was uniform and in line with the characteristics of C-type BSE.


Biochemical typing of pathological prion protein in aging cattle with BSE.

Tester S, Juillerat V, Doherr MG, Haase B, Polak M, Ehrensperger F, Leeb T, Zurbriggen A, Seuberlich T - Virol. J. (2009)

Biochemical PrPres typing in diagnostic target sites of aged cattle with BSE. PrPres was analyzed at least five times on different gels with a core-binding antibody (mAb 6H4) by Western blot. a) Average molecular masses of the unglycosylated (red open circles), the monoglycosylated (green filled circles) and the diglycosylated band (blue rectangles) of PrPres are depicted with the related standard errors of the mean (S.E.M.). The cut-off value for the molecular mass of the unglycosylated band to discriminate H-type from C-type BSE was defined as the average molecular mass of all samples under investigation (solid line) +/- 5% (dashed line). b) Tri-plot graph presenting the relative intensities of the un-, mono- and diglycosylated PrPres moieties. The L-type BSE control (green square) is the sole sample that fell below the decision limit of 55% relative intensity of the diglycosylated PrPres (blue dashed line). The H-type BSE control is indicated by a red triangle and the zebu Charly-04 by a yellow circle. Samples were derived from medulla oblongata at the level of obex but where this was not available hippocampus was examined (marked by asterisks). CS, clinical suspect; ES, emergency slaughter; FS, fallen stock; RS, routine slaughter.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2693104&req=5

Figure 2: Biochemical PrPres typing in diagnostic target sites of aged cattle with BSE. PrPres was analyzed at least five times on different gels with a core-binding antibody (mAb 6H4) by Western blot. a) Average molecular masses of the unglycosylated (red open circles), the monoglycosylated (green filled circles) and the diglycosylated band (blue rectangles) of PrPres are depicted with the related standard errors of the mean (S.E.M.). The cut-off value for the molecular mass of the unglycosylated band to discriminate H-type from C-type BSE was defined as the average molecular mass of all samples under investigation (solid line) +/- 5% (dashed line). b) Tri-plot graph presenting the relative intensities of the un-, mono- and diglycosylated PrPres moieties. The L-type BSE control (green square) is the sole sample that fell below the decision limit of 55% relative intensity of the diglycosylated PrPres (blue dashed line). The H-type BSE control is indicated by a red triangle and the zebu Charly-04 by a yellow circle. Samples were derived from medulla oblongata at the level of obex but where this was not available hippocampus was examined (marked by asterisks). CS, clinical suspect; ES, emergency slaughter; FS, fallen stock; RS, routine slaughter.
Mentions: To investigate the PrPres phenotype in detail we initially analyzed samples from the medulla oblongata (or from the hippocampus when MO was not available) in WB protocol I and we determined the molecular masses and relative proportions of the PrPres glycoforms. Only the H-type control and the H-type zebu revealed an unglycosylated PrPres band of an average molecular mass above 19 kDa with mAb 6H4. All other animals including the L-type control showed an average molecular mass in the range of 17.5 kDa to 18.82 kDa, which lies within the decision limit of ± 5% of the overall average molecular mass (Figure 2a). The average relative intensity of the diglycosylated band in all aging cattle with BSE was above the decision limit of 55% (57.5% to 64.9%) and for the L-type, as expected, much lower at 49.9% (Figure 2b). None of the cattle samples showed considerable reactivity with the N-terminal mAb 12B2 and all displayed a three-band pattern with mAb SAF84 (data not shown). Hence, the PrPres phenotype of the remaining 32 Swiss cattle included in this study was uniform and in line with the characteristics of C-type BSE.

Bottom Line: Our work confirmed H-type BSE in a zebu but classified all other cases as C-type BSE; indicating a very low incidence of H- and L-type BSE in Switzerland.It was documented for the first time that the biochemical PrPres type was consistent across different brain regions of aging animals with C-type and H-type BSE, i.e. independent of the neuroanatomical structure investigated.Taken together this study provides further characteristics of the BSE epidemic in Switzerland and generates new baseline data for the definition of C- and H-type BSE phenotypes, thereby underpinning the notion that they indeed represent distinct prion disease entities.

View Article: PubMed Central - HTML - PubMed

Affiliation: NeuroCenter, Reference Laboratory for TSE in animals, Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Berne, Switzerland. seraina.tester@itn.unibe.ch

ABSTRACT

Background: The broad enforcement of active surveillance for bovine spongiform encephalopathy (BSE) in 2000 led to the discovery of previously unnoticed, atypical BSE phenotypes in aged cattle that differed from classical BSE (C-type) in biochemical properties of the pathological prion protein. Depending on the molecular mass and the degree of glycosylation of its proteinase K resistant core fragment (PrPres), mainly determined in samples derived from the medulla oblongata, these atypical cases are currently classified into low (L)-type or high (H)-type BSE. In the present study we address the question to what extent such atypical BSE cases are part of the BSE epidemic in Switzerland.

Results: To this end we analyzed the biochemical PrPres type by Western blot in a total of 33 BSE cases in cattle with a minimum age of eight years, targeting up to ten different brain regions. Our work confirmed H-type BSE in a zebu but classified all other cases as C-type BSE; indicating a very low incidence of H- and L-type BSE in Switzerland. It was documented for the first time that the biochemical PrPres type was consistent across different brain regions of aging animals with C-type and H-type BSE, i.e. independent of the neuroanatomical structure investigated.

Conclusion: Taken together this study provides further characteristics of the BSE epidemic in Switzerland and generates new baseline data for the definition of C- and H-type BSE phenotypes, thereby underpinning the notion that they indeed represent distinct prion disease entities.

Show MeSH
Related in: MedlinePlus