Biological and biochemical characterization of L-type-like bovine spongiform encephalopathy (BSE) detected in Japanese black beef cattle

Abstract

A case of L-type-like atypical bovine spongiform encephalopathy was detected in 14-year-old Japanese black beef cattle (BSE/JP24).  To clarify the biological and biochemical properties of the prion in BSE/JP24, we performed a transmission study with wild-type mice and bovinized transgenic mice (TgBoPrP).  The BSE/JP24 prion was transmitted to TgBoPrP mice with the incubation period of 199.7 ± 3.4 days, which was shorter than that of classical BSE (C-BSE) (223.5 ± 13.5 days).  Further, C-BSE was transmitted to wild-type mice with the incubation period of about 409 days, whereas BSE/JP24 prion inoculated mice showed no clinical signs up to 649 days.  Severe vacuolation and a widespread and uniform distribution of PrPSc were pathologically observed in the brain of BSE/JP24 prion affected TgBoPrP mice.  The molecular weight and glycoform ratio of PrPSc in BSE/JP24 were different from those in C-BSE, and PrPSc in BSE/JP24 exhibited weaker proteinase K resistance than that in C-BSE.  These findings revealed that the BSE/JP24 prion has distinct biological and biochemical properties reported for that of C-BSE.  Interestingly, a shorter incubation period was observed at the subsequent passage of the BSE/JP24 prion to TgBoPrP mice (152.2 ± 3.1 days).  This result implies that BSE/JP24 prion has newly emerged and showed the possibility that L-type BSE prion might be classified into multiple strains.

Acknowledgements

We thank Ms. Naoko Tabeta, Shuko Kodani, Nozomi Matsumoto, Atsuko Ojima, Ritsuko Miwa and Mutsumi Sakurai for their technical assistance. Further, we thank Dr. Morikazu Shinagawa for his encouragement; Ms. Junko Yamada for her general assistance; and Mr. Manabu Aida, Ms. Chizuru Kuramochi, Che Jing Zh and the animal laboratory staff at the National Institute of Animal Health for maintaining the mouse colony. This study was supported in part by a Grant-in-Aid from the Bovine Spongiform Encephalopathy Control Project of the Ministry of Agriculture, Forestry and Fisheries of Japan; a grant for BSE research from the Ministry of Health, Labour and Welfare of Japan; and a grant-in-aid for the strategic cooperation to control emerging and reemerging infection from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Figures and Tables

Figure 1 Western blotting analysis of PrP^Sc from C-BSE and BSE/JP24. (A) The fragment of PrP^Sc in the cattle brain of C-BSE (lanes 1 and 3) and BSE/JP24 (lanes 2 and 4). PrP^Sc in the brain of TgBoPrP inoculated with C-BSE prion (lanes 5 and 7) and BSE/JP24 prion (lanes 6 and 8). All samples were digested with 50 µg/ml PK at 37°C for 1 h, and then samples in lanes 3, 4, 7 and 8 were treated with PNGaseF. PrP^Sc was detected by using mAb 6H4. Molecular markers are shown on the left (kDa). (B) The relative amount of the di-, mono- and non-glycosylated form of PrP^Sc in the C-BSE and BSE/JP24 prion affected individual. The results are mean ± standard deviation in five experiments. Bar diagram: diglycosylated form (black), monoglycosylated form (grey) and nonglycosylated form (white).

Figure 2 Lesion profile of TgBoPrP mice inoculated with C-BSE and BSE/JP24 prions. Vacuolation was scored on a 0–5 (mean values) in the following brain areas: 1, dorsal medulla; 2, cerebellar cortex; 3, superior cortex; 4, hypothalamus; 5, thalamus; 6, hippocampus; 7, septal nuclei of the paraterminal body; 8, cerebral cortex at the levels of 4 and 5; and 9, cerebral cortex at the level of 7. •: 1^st passage of BSE/JP24 prion, ○: 2^nd passage of BSE/JP24 prion, ▴: 1^st passage of C-BSE prion.

Figure 3 Histopathological (A and B), immunohistochemical (C and D) and PET-blot (E and F) analysis of TgBoPrP mice inoculated with C-BSE and BSE/JP24 prions. No distinct vacuolation in the presence of PrP plaques was detected in the cerebral cortex and hippocampal region in C-BSE prion affected TgBoPrP mice (A), whereas severe vacuolation in the absence of PrP-positive deposits was prominent in BSE/JP24 prion affected TgBoPrP mice (B). Immunolabelled PrP^Sc showed coarse granular and coalescing-like patterns in the gigantocellular nucleus of medulla oblongata of C-BSE prion affected TgBoPrP mice (C). A diffused fine granular pattern was observed in BSE/JP24 prion affected TgBoPrP mice (D). Immunohistochemical labelling with mAb F99/97.6.1. The insets at the lower right corner (A and C) are PrP plaques detected in the periventricular area of frontal lobe (arrowheads). PET blot reveals that immunolabelled PrP^Sc was marked in particular nuclei of brainstems in C-BSE prion affected TgBoPrP mice (E). On the other hand, widespread and homogeneous PrP^Sc immunolabelling was obvious in BSE/JP24 prion affected TgBoPrP mice (F). PET blots of the representative coronal section at the level of the hippocampus (left) and medulla oblongate (right) are shown. The mAb SAF84 was used in PET-blot analysis. Bar: 200 µm (A and B); 50 µm (C and D); 20 µm (inset of A and C).

Figure 4 Relative PK resistance of PrP^Sc in prion affected cattle and TgBoPrP mice. (A) PK resistance of PrP^Sc in the cattle brain of C-BSE and BSE/JP24. (B) PK resistance of PrP^Sc of TgBoPrP mice inoculated with C-BSE and BSE/JP24 prions. (C) PK resistance of PrP^Sc from the subsequent passage of C-BSE and BSE/JP24 prion to TgBoPrP mice. The PrP^Sc concentration of sample was adjusted by the signal intensity of western blotting. The samples were treated with PK of various concentration (40–1,000 µg/ml) at 37°C for 1 h. Data shown represent one of three experiments demonstrating similar trends. PrP^Sc was detected with mAb 6H4. Molecular markers are shown on the left (kDa).

Table 1 Transmission of C-BSE and BSE/JP24 prion to mice

Table 2 Conformational stability ([GdnHCl]_1/2 value) of PrP^Sc from C-BSE and BSE/JP24