Table 1.
Abnormal PrP (PrPSc) and infectivity in central nervous system and lympho-reticular system of natural Atypical/Nor98 scrapie incubating or affected animals bearing various genotypes at codons 136, 141, 154 and 171 of the Prnp gene.
Figure 1.
PrPSc Western Blot detection in sheep and tg338 mice inoculated with Atypical/Nor98 scrapie and classical scrapie tissues (10% tissue homogenate).
Lane 1: posterior brainstem from a Langlade classical scrapie affected sheep (case 10). Lane 2: brain from a tg338 mouse inoculated with striated muscle (semi-membranous) from the same sheep (case 10). Lane 3: posterior brainstem from a PG127 classical scrapie orally inoculated sheep (case 12). Lane 4: brain from a tg338 mouse inoculated with striated muscle (extra-ocular motor muscle) from the same PG127 classical scrapie affected sheep (case 12). Lane 5: cerebral cortex from an AFRQ/AFRQ Atypical/Nor98 scrapie natural case (case 1). Lane 6: AHQ/AHQ sheep (cerebellum-case 9) intra-cerebrally challenged with natural atypical scrapie (case 1). Lane 7 to 9: brain homogenates from tg338 mice inoculated with peripheral tissues from atypical scrapie cases. Lane 7: retropharyngeal lymph node (case 2). Lane 8: sciatic nerve (case 9). Lane 9: striated muscle (case 8). No PrPSc was observed in control tg338 mice inoculated with retropharyngeal LN from a negative control sheep (Lane 10).
Figure 2.
PrPSc distribution pattern and lesion profile in tg338 mice inoculated with Atypical/Nor98 scrapie and classical scrapie sheep tissues.
(A–D) PET Blot (SHa31 antibody – NBT/BCIP black deposits- bar: 200 µm) of brain coronal section (thalamus level) from tg338 inoculated with (A) AHQ/AHQ atypical scrapie cerebellum (case 9), (B) posterior brainstem from VRQ/VRQ classical scrapie (PG127- case 12), (C) retropharyngeal lymph node from a ARR/ARR natural atypical case at preclinical stage of the disease (case 3), (D) striated muscle from a AFRQ/ARQ experimental atypical case (case 8) at clinical stage of the disease. (E–H) Lesion profile (vacuolar changes) in tg338 mice inoculated with: (E) Natural (case 3 - △) and experimental (case 9- ●) cerebral cortex from atypical scrapie cases, (F) Langlade (case 10-▽) and PG127 (case 12- ●) VRQ/VRQ posterior brainstem homogenates, (G) brachial nerve from an AFRQ/ARQ experimental atypical case (case 8-◆) and AFRQ/AFRQ natural atypical case (case 1-▽), (H) striated muscle from Langlade (case 10- ▽- semi-membranous) and PG127 (case 12- ●-psoas) classical scrapie affected sheep.
Table 2.
PrPSc and infectivity in tissues from experimental (IC challenged) Atypical/Nor98 scrapie cases and natural or experimental (oral or intra-cerebral challenge) classical scrapie cases.
Figure 3.
PrPSc detection limit of a Langlade classical scrapie isolate, a PG127 classical scrapie isolate and an Atypical/Nor98 scrapie isolate.
Dilution series from (A, D) Atypical/Nor98 scrapie isolate (case 9- cerebral cortex), (B) Langlade–case 10- posterior brainstem) and (C) PG127 (case 12- posterior brainstem) classical scrapie isolate, that were prepared in negative sheep brain homogenate. The tissue homogenates (see methods) are the same than the one used for endpoint titration in tg338 mice (Table 3). The samples were processed for PrPSc Western-blotting (A–C, TeSeE WB kit – BioRad, anti-PrP SHa31 antibody). After extraction (25 mg of brain equivalent material), the re-suspended pellet was either entirely loaded on lanes (A: all lanes – B and C: Lanes 4–6) or diluted in Laemmli's buffer before loading (B: lane 3, 1/50 – Lane 4, 1/10 – C: lane 3, 1/20). These dilutions were necessary to avoid a saturation of the signal. A classical scrapie control was included on the three gels to calibrate the signal (Lane 1). (D) The atypical/Nor98 scrapie isolate dilution serie (case 9: cerebral cortex) was tested using a commercially available rapid TSE test (TeSeE Sheep and Goat - BioRad) used for field TSE screening in small ruminants. Three different aliquots of each dilution were independently tested. The extractions were carried out. Results are presented as the mean +/− SD corrected optical density values. The cut off value (0.162 OD– dotted lines) was established as the mean of four negative control optical densities + 0.150 OD.
Table 3.
Classical scrapie and Atypical/Nor98 scrapie isolates endpoint titration and infectious titre in ovine PrP transgenic mice (tg338).
Figure 4.
Infectivity titre in reference brain sample from classical and Atypical/Nor98 affected ewes.
(A–B) Intra-cerebral endpoint titration in a tg338 mouse model of CNS homogenate (20 µl per mice), (A) prepared from terminally scrapie affected sheep with Langlade isolate (12.5% homogenate - case 10 - ○) and with PG127 isolate (10% homogenate- case 13- △) – (B) an ARQ/ARQ (10% homogenate - case 14- ○) and an ARR/ARR (10% homogenate -case 15- △) atypical scrapie cases identified in field through active epidemiosurveillance program. Such titration allowed the determination of the infectious dose 50 (ID50) of the reference brain samples (Table 3). (D–E) Variation of the incubation period as a function of the infectious dose inoculated intra-cerebrally in tg338 mice. (C): Langlade isolate, (D): PG127 isolate and (E): atypical scrapie isolate. To establish these diagrams, the individual incubation periods and the number of ID50 inoculated in each mouse were plotted. The number of ID50 is obtained by multiplying the titre of the inoculum (ID50 IC in tg338 per gram of tissue) per the amount of tissue inoculated in each mice. On this basis, a four parameter logistic regression was computed (Sigma Plot) to provide a curve associating the incubation period and the number of ID50 inoculated with the observed incubation period.