Intraspecies prion transmission results in selection of sheep scrapie strains

Abstract

Background: Sheep scrapie is caused by multiple prion strains, which have been classified on the basis of their biological characteristics in inbred mice. The heterogeneity of natural scrapie prions in individual sheep and in sheep flocks has not been clearly defined.

Methodology/principal findings: In this study, we intravenously injected 2 sheep (Suffolk and Corriedale) with material from a natural case of sheep scrapie (Suffolk breed). These 3 sheep had identical prion protein (PrP) genotypes. The protease-resistant core of PrP (PrPres) in the experimental Suffolk sheep was similar to that in the original Suffolk sheep. In contrast, PrPres in the Corriedale sheep differed from the original PrPres but resembled the unusual scrapie isolate, CH1641. This unusual PrPres was not detected in the original sheep. The PrPres distributions in the brain and peripheral tissues differed between the 2 breeds of challenged sheep. A transmission study in wild-type and TgBoPrP mice, which overexpressing bovine PrP, led to the selection of different prion strains. The pathological features of prion diseases are thought to depend on the dominantly propagated strain.

Conclusions/significance: Our results indicate that prion strain selection occurs after both inter- and intraspecies transmission. The unusual scrapie prion was a hidden or an unexpressed component in typical sheep scrapie.

Figure 1. Western blotting analysis of PrPres in scrapie sheep brain.

Obex homogenates were subjected to analysis by Western blot. Each lane contained 0.5 mg sheep brain equivalent sample. Lane 1: G3571, lane 2: #2314 (G3571-inoculated Suffolk sheep), lane 3: #294 (G3571-inoculated Corriedale sheep), Mo: mouse-adapted scrapie Obihiro (25 µg brain equivalent), b: classical natural BSE (C-BSE) (0.5 mg brain equivalent). A and D. PrPres was detected using mab T2. PrPres in lanes 1, and 2 was classified as high-molecular-weight PrPres (h-type PrPres), and that in lanes 3 was classified as low-molecular-weight PrPres (l-type PrPres). B and E. PrPres was detected using mab SAF-84. A 14-kDa fragment of PrPres (PrPres #2) was detected in #294. C. PrPres was detected using mab P4. PrPres was analyzed before (A, B and C) or after (D and E) PNGase F deglycosylation. Size markers (in kDaltons) are indicated on the left.

Figure 2. Glycoform profile of PrPres in the sheep brain.

Glycoforms of PrPres that accumulated in the experimentally scrapie-challenged sheep were analyzed. We analyzed the signal intensity of the bands shown in Fig. 1A. PrPres was detected by mab T2. Black bar: Ratio of diglycosylated PrPres to total PrPres. Dashed bar: Monoglycosylated PrPres. White bar: Non-glycosylated PrPres. Values are expressed as the mean and standard deviation (percentage).

Figure 3. PrPres detection from different regions of the sheep brain.

Each lane contained 0.5 mg sheep brain equivalent sample. Lane 1: cerebral cortex, lane 2: brainstem (pons), lane 3: cerebellar medulla, lane 4: cerebellar cortex, lane 5: obex, Mo: mouse-adapted scrapie (25 µg brain equivalent). b: C-BSE (0.5 mg brain equivalent), h: H-type atypical BSE (0.5 mg brain equivalent). PrPres from (A) #2314 (Suffolk sheep) and (B) #294 (Corriedale sheep) was detected using mab T2. C. PrPres from #294 was detected by mab SAF-84.

Figure 4. Analysis by neuropathology of experimentally scrapie-challenged sheep.

Immunohistochemical analysis: a. frontal cortex, #2314; b. cerebellum, #2314; c. frontal cortex, #294; and d. cerebellum, #294. PrP^Sc immunolabeling was performed using mab T1. Bar: 100 µm.

Figure 5. PrPres profile in the nervous and lymphoid tissues of #294 (Corriedale sheep).

Lane 1: anterior cervical lymph node, lane 2: mesenteric lymph node, 3: optic nerve, and 4: brainstem. b: C-BSE, h: H-type atypical BSE. A. PrPres was detected using mab T2. In #294, l-type PrPres was detected from the optic nerve and brainstem, whereas h-type PrPres was detected from the lymphoid tissues. B. PrPres was also detected using mab SAF-84. An additional 14-kDa band was detected from l-type PrPres, but not from h-type PrPres.

Figure 6. PrPres analysis in scrapie-passaged mice.

PrPres that accumulated in the brains of scrapie-passaged mice was analyzed by Western blot. Lanes 1 and 2: C-BSE-passaged mice, lanes 3 and 4: G3571-passaged mice, lanes 5 and 6: mice passaged with samples from #2314 (Suffolk), lane 7: mice passaged with samples from #294 (Corriedale). Lanes 1, 3, 5: ICR mice. Lanes 2, 4, 6 and 7: TgBoPrP mice. Mo: mouse-adapted scrapie. PrPres was detected using mab SAF-84.

Figure 7. Illustrative models of dominant PrPres in the brains of sheep and mice.

PrPres was classified on the basis of its molecular weight (h-type or l-type) and the existence of the PrPres #2 fragment. The dashed lines indicate similar PrPres. In the case of sheep transmission, PrPres in #2314 was similar to that in G3571. In the case of mouse transmission, similar PrPres was amplified in wild-type mice inoculated with samples from #2314 and G3571. All the 3 scrapie-passaged TgBoPrP mice harbored similar PrPres in their brains. Prions from #294 were not transmitted to wild-type mice. The mean incubation period (days) is indicated beside the arrows. WT mice: wild-type mice.