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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 typing of different brain regions in H-type BSE. Western blot analysis of the H-type BSE zebu (Charly-04) with a) a core-binding antibody (Sha31), b) an amino-terminal binding antibody (12B2) and c) a carboxy-terminal binding antibody (SAF84). Samples are assigned to the lanes as follows: negative control (N), L-type BSE (L), C-type BSE (C) and for the zebu medulla oblongata (lane 1, 15 mg tissue equivalent), cerebellar cortex (lane 2, 15 mg), hippocampus (lane 4, 0.75 mg), piriform lobe (lane 5, 15 mg), basal ganglia (lane 7, 1.5 mg), frontal cortex (lane 8, 15 mg), occipital cortex (lane 9, 15 mg) and temporal cortex (lane 10, 15 mg). The dashed line indicates the molecular mass of the unglycosylated C-type PrPres and helps to visualize differences compared to the H-type BSE zebu. The same samples, but deglycosylated are shown in d) with a carboxy-terminal binding antibody (SAF84). A molecular mass marker (in kDa) is indicated on the left.
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Figure 5: Biochemical typing of different brain regions in H-type BSE. Western blot analysis of the H-type BSE zebu (Charly-04) with a) a core-binding antibody (Sha31), b) an amino-terminal binding antibody (12B2) and c) a carboxy-terminal binding antibody (SAF84). Samples are assigned to the lanes as follows: negative control (N), L-type BSE (L), C-type BSE (C) and for the zebu medulla oblongata (lane 1, 15 mg tissue equivalent), cerebellar cortex (lane 2, 15 mg), hippocampus (lane 4, 0.75 mg), piriform lobe (lane 5, 15 mg), basal ganglia (lane 7, 1.5 mg), frontal cortex (lane 8, 15 mg), occipital cortex (lane 9, 15 mg) and temporal cortex (lane 10, 15 mg). The dashed line indicates the molecular mass of the unglycosylated C-type PrPres and helps to visualize differences compared to the H-type BSE zebu. The same samples, but deglycosylated are shown in d) with a carboxy-terminal binding antibody (SAF84). A molecular mass marker (in kDa) is indicated on the left.

Mentions: As we had access to brain tissues apart from the medulla oblongata in a series of the cattle with C-type and the zebu with H-type BSE (table 1), we analyzed for the consistency of the biochemical phenotype within these animals in up to ten different neuroanatomical structures per case. Regarding the molecular mass and the glycoform proportions (for examples see Figure 3 and for the complete data see Additional file 1) as well as the reactivity with mAbs 12B2 and SAF84 all cattle samples showed the characteristics of C-type BSE in all the brain structures (data not shown). Occasionally we observed a second band slightly above the unglycosylated one with mAb 6H4. However, this was not reproducible when we repeated the analysis starting from the same homogenate and resulted then in a pattern indicative for C-type BSE. In two others (Bunaug-02, MO, Figure 2; Martina-96, TH; Figure 3) the average molecular mass of the unglycosylated band was near to the upper decision limit. We decided to retest these samples in an alternative WB (WB protocol II) using mAb Sha31 as the core-specific antibody and a different PK digestion procedure. Here, both samples clearly revealed a molecular mass similar to C-type and not to H-type BSE (Figure 4). We also investigated the PrPres phenotype in eight different brain regions of the H-type BSE affected zebu. Remarkably, all three criteria to identify H-type BSE, i.e. the higher molecular mass of unglycosylated PrPres with the core antibody Sha31 (Figure 5a), the reactivity with mAb 12B2 (Figure 5b) and the complex banding pattern with mAb SAF84 (Figure 5c) were consistently fulfilled in the zebu irrespective of the neuroanatomical structure. After deglycosylation with PNGase F all brain regions investigated in the zebu revealed two bands at ~20.0 kDa and ~14.0 kDa while in C- and L-type BSE only one band at 18.5 kDa to 19.5 kDa could be identified with the C-terminally-binding mAb SAF84 (Figure 5d).


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 typing of different brain regions in H-type BSE. Western blot analysis of the H-type BSE zebu (Charly-04) with a) a core-binding antibody (Sha31), b) an amino-terminal binding antibody (12B2) and c) a carboxy-terminal binding antibody (SAF84). Samples are assigned to the lanes as follows: negative control (N), L-type BSE (L), C-type BSE (C) and for the zebu medulla oblongata (lane 1, 15 mg tissue equivalent), cerebellar cortex (lane 2, 15 mg), hippocampus (lane 4, 0.75 mg), piriform lobe (lane 5, 15 mg), basal ganglia (lane 7, 1.5 mg), frontal cortex (lane 8, 15 mg), occipital cortex (lane 9, 15 mg) and temporal cortex (lane 10, 15 mg). The dashed line indicates the molecular mass of the unglycosylated C-type PrPres and helps to visualize differences compared to the H-type BSE zebu. The same samples, but deglycosylated are shown in d) with a carboxy-terminal binding antibody (SAF84). A molecular mass marker (in kDa) is indicated on the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Biochemical typing of different brain regions in H-type BSE. Western blot analysis of the H-type BSE zebu (Charly-04) with a) a core-binding antibody (Sha31), b) an amino-terminal binding antibody (12B2) and c) a carboxy-terminal binding antibody (SAF84). Samples are assigned to the lanes as follows: negative control (N), L-type BSE (L), C-type BSE (C) and for the zebu medulla oblongata (lane 1, 15 mg tissue equivalent), cerebellar cortex (lane 2, 15 mg), hippocampus (lane 4, 0.75 mg), piriform lobe (lane 5, 15 mg), basal ganglia (lane 7, 1.5 mg), frontal cortex (lane 8, 15 mg), occipital cortex (lane 9, 15 mg) and temporal cortex (lane 10, 15 mg). The dashed line indicates the molecular mass of the unglycosylated C-type PrPres and helps to visualize differences compared to the H-type BSE zebu. The same samples, but deglycosylated are shown in d) with a carboxy-terminal binding antibody (SAF84). A molecular mass marker (in kDa) is indicated on the left.
Mentions: As we had access to brain tissues apart from the medulla oblongata in a series of the cattle with C-type and the zebu with H-type BSE (table 1), we analyzed for the consistency of the biochemical phenotype within these animals in up to ten different neuroanatomical structures per case. Regarding the molecular mass and the glycoform proportions (for examples see Figure 3 and for the complete data see Additional file 1) as well as the reactivity with mAbs 12B2 and SAF84 all cattle samples showed the characteristics of C-type BSE in all the brain structures (data not shown). Occasionally we observed a second band slightly above the unglycosylated one with mAb 6H4. However, this was not reproducible when we repeated the analysis starting from the same homogenate and resulted then in a pattern indicative for C-type BSE. In two others (Bunaug-02, MO, Figure 2; Martina-96, TH; Figure 3) the average molecular mass of the unglycosylated band was near to the upper decision limit. We decided to retest these samples in an alternative WB (WB protocol II) using mAb Sha31 as the core-specific antibody and a different PK digestion procedure. Here, both samples clearly revealed a molecular mass similar to C-type and not to H-type BSE (Figure 4). We also investigated the PrPres phenotype in eight different brain regions of the H-type BSE affected zebu. Remarkably, all three criteria to identify H-type BSE, i.e. the higher molecular mass of unglycosylated PrPres with the core antibody Sha31 (Figure 5a), the reactivity with mAb 12B2 (Figure 5b) and the complex banding pattern with mAb SAF84 (Figure 5c) were consistently fulfilled in the zebu irrespective of the neuroanatomical structure. After deglycosylation with PNGase F all brain regions investigated in the zebu revealed two bands at ~20.0 kDa and ~14.0 kDa while in C- and L-type BSE only one band at 18.5 kDa to 19.5 kDa could be identified with the C-terminally-binding mAb SAF84 (Figure 5d).

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