Figures & data
Table 1. Clinical/neurological examination observations for the three oral challenge C type BSE steers. The clinical signs started at 45 months post challenge and progressed over the next six months. All three steers displayed similar and significant clinical signs of neurologic disease at 52 months post challenge and were sacrificed
Table 2. Molecular diagnostic test results for the three cBSE experimental steers as well as a negative and positive control animal. The positive/negative cut-off is listed for the Priostrip and IDEXX ELISA runs. PTA WB (phosphotungstic acid western blot) is visually interpreted, +++ is indicative of three very strong immunoreactive bands (one for each glycoform) that are difficult to separate due to the intensity of the signal
Figure 1. A) Western blot immunodetection of PK resistant prion proteins in the brain samples of the 3 oral challenge steers. Exp. cBSE 1 (29,014) and Exp. cBSE 2 (29,055) both have a strong positive signal with 3 immunoreactive bands at 30kDa and below. Exp. cBSE 3 (29,034) has no immunoreactivity in western blot. Western blot immunodetection of PK resistant PMCA products in different dilutions of brain homogenate used to spike into the PMCA reactions. b) Round 3 PMCA seeding activity/PK resistant PrP is detected in low dilutions for Exp. cBSE 1 (29,014) and 2 (29,055) indicating a high concentration of seeding units. Exp. cBSE 3 (29,034) has much less seeding activity
Figure 2. IHC and PMCA results from the Tg Bov XV mouse brains after being challenged intra-cranially with Exp. cBSE 1 (29,014) and Exp. cBSE 3 (29,034). a) Immunohistochemical staining of prion protein aggregates using anti-prion antibody F99. Some non-specific staining is seen around blood vessels but no aggregates are detected. b) Prion protein aggregates are detected in a number of areas in this transgenic mouse brain (arrows). c) Western blot detection of PK resistant prion proteins indicative of seeding activity in the brains of the mice tested. Mice challenged with Exp. cBSE 1 seeded PMCA formation of PK resistant PrP in all rounds (Sample 15). None of the Exp. cBSE 3 challenged mouse brains seeded PMCA conversion of PrP to PrPSc indicating no transmission of seeding activity from the index animal. Samples 1–12 3927 to 3938 (Exp. cBSE 3 (29,034) challenged mice), Sample 13: 3947 (29,059(BSE-), 595dpi), Sample 14: 3948 (29,059(-), 596dpi), Sample 15: 3940 (29,014(BSE+), 299dpi)
Figure 3. Nucleotide base sequence alignment and amino acid translation of the 3 oral cBSE challenged steers. Exp. cBSE 1 (29,014) grey text, Exp. cBSE 2 (29,055) black text, Exp. cBSE 3 (29,034) red text. Only 2 single nucleotide polymorphisms were identified in Exp. cBSE 1 but both are silent and do not result in an amino acid change
Figure 4. Variable SNPs in the coding and noncoding region of the PRNP gene of the 3 experimental BSE steers and 16 of the Canadian BSE field cases. SNP comparisons and numbering are based on PubMed reference sequence AJ298878. SNPs that are homozygous different than the reference sequence are shaded in dark grey, heterozygous SNPs are light grey and homozygous SNPs matching the reference sequence are unshaded. *23bp indel, **12bp indel, ***unstudied indel site, ~coding sequence SNPs
Table 3. Fraction of the breed composition that is Hereford for the three experimental BSE steers and 16 of the Canadian BSE field cases and the BSE result for each of these animals. Composition Hereford is a fraction out of a total of 1.0