Sheep and goat BSE propagate more efficiently than cattle BSE in human PrP transgenic mice

Abstract

A new variant of Creutzfeldt Jacob Disease (vCJD) was identified in humans and linked to the consumption of Bovine Spongiform Encephalopathy (BSE)-infected meat products. Recycling of ruminant tissue in meat and bone meal (MBM) has been proposed as origin of the BSE epidemic. During this epidemic, sheep and goats have been exposed to BSE-contaminated MBM. It is well known that sheep can be experimentally infected with BSE and two field BSE-like cases have been reported in goats. In this work we evaluated the human susceptibility to small ruminants-passaged BSE prions by inoculating two different transgenic mouse lines expressing the methionine (Met) allele of human PrP at codon 129 (tg650 and tg340) with several sheep and goat BSE isolates and compared their transmission characteristics with those of cattle BSE. While the molecular and neuropathological transmission features were undistinguishable and similar to those obtained after transmission of vCJD in both transgenic mouse lines, sheep and goat BSE isolates showed higher transmission efficiency on serial passaging compared to cattle BSE. We found that this higher transmission efficiency was strongly influenced by the ovine PrP sequence, rather than by other host species-specific factors. Although extrapolation of results from prion transmission studies by using transgenic mice has to be done very carefully, especially when human susceptibility to prions is analyzed, our results clearly indicate that Met129 homozygous individuals might be susceptible to a sheep or goat BSE agent at a higher degree than to cattle BSE, and that these agents might transmit with molecular and neuropathological properties indistinguishable from those of vCJD. Our results suggest that the possibility of a small ruminant BSE prion as vCJD causal agent could not be ruled out, and that the risk for humans of a potential goat and/or sheep BSE agent should not be underestimated.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1. Comparative PrPres content of the cattle, sheep and goat isolates used for tgHu bioassays.

Immunoblots of brain PrPres detected with SHA 31 mAb. Direct sample and 1/5 dilutions were loaded on 12% Bis-Tris gels. Molecular weights (in kDa) are shown at the left side of the blot. Excluding the BSE0 isolate, all cattle BSE isolates presented higher PrPres contents than the sheep and goat BSE isolates. Cattle BSE2 and BSE3 isolates presented comparable PrPres contents while that in the BSE0 isolate was substantially lower. The sheep and goat BSE isolates used contained similar PrPres quantity.

Figure 2. Western blots analysis of PrPres in the brains of tg650 mice infected with human, bovine, ovine and goat isolates.

Immunoblots of brain PrPres detected with SHA 31 Mab. Similar amount of brain tissue equivalent (20 µg) was loaded for adequate comparison. vCJD isolate was loaded for comparison. Molecular weights (in kDa) are shown at the left side of the blot. Similar PrPres banding patterns were observed in the vCJD- and the cattle, sheep and goat BSE-challenged tg650 mice.

Figure 3. Western blots analysis of PrPres in the brains of tg340 mice infected with human, bovine, ovine and goat isolates.

Immunoblots of brain PrPres detected with SHA 31 Mab. Similar quantities of PrPres were loaded for adequate comparison. sCJD and vCJD1 isolates were loaded for comparison. Molecular weights (in kDa) are shown at the left side of the blot. Note the typical PrPvCJD banding pattern, characterized by low size fragments and prominent diglycosylated species in the cattle, sheep and goat BSE-challenged mice. These banding patterns clearly differed from that observed after inoculation of mice with sporadic CJD.

Figure 4. Western blots analysis of PrPres in the brains of tg340 mice infected with human, bovine and ovine isolates.

Immunoblots of brain PrPres detected with SHA 31 and 12B2 mAbs. Similar quantities of PrPres were loaded for adequate comparison. sCJD and vCJD1 isolates were loaded for comparison. Molecular weights (in kDa) are shown at the left side of the blot. Both vCJD- and type I sCJD-like PrPres were observed after second passage of Ca-BSE2 in tg340 mice (Ca-BSE2/Tg340 2P-b and Ca-BSE2/Tg340 2P-a respectively).

Figure 5. Regional distribution of PrPres in the brain of human PrP transgenic mice infected with BSE agent passaged through sheep, goat or human species.

Histoblots of 4 representative anteroposterior mouse brain sections are shown. Comparison has been made at 3rd passage in tg650 mice. The patterns produced by sheep, goat and cattle BSE and variant CJD appear similar.

Figure 6. Brain vacuolar changes and amyloid plaques in transgenic mice for the human PRNP gene (methionine 129 variant - tg650).

Hemalun-Eosin staining. Mice were inoculated with natural goat BSE (a – Bar 150 µm), cattle BSE (b: bar150 µm-e: bar 50 µm), vCJD (c: bar 150 µm-d: bar 50 µm) or experimental BSE in sheep (ARQ/ARQ genotype) (f: bar 50 µm). Vacuolar changes are associates to plaques deposits (a–b–c) (in a *: plaques deposits). Plaques deposits often harboured typical features of florid plaques: presence of crown of vacuoles and glial cell nuclei (d–e–f).

Figure 7. Transmission to human PrP mice of different BSE isolates generated by intermediary passages in either ovine or bovine PrP transgenic mice.

na: not available (experiments still ongoing). aIntracerebral inoculation with 2 mg brain tissue equivalent; n/n0: diseased, PrPres positive/inoculated animals. cTwo serial passages on tgBov mice.

Figure 8. Brain PrPC expression in homozygous tg340 mouse line in comparison to both tg650 mice and human brain.

A) Immunoblots of the brain PrPC expression in tg340 detected with Pri 308 Mab. Direct sample (10% brain homogenates) and ¼ dilutions were loaded on 12% Bis-Tris gels. The figure illustrates a representative set of three independent experiments. B) Brain PrPC expression in tg340 in comparison to tg650 detected with Pri 308 Mab. Inmunoblots illustrates a representative set of three independent experiments and the diagrams represent the mean densytometric values data from these experiments. Data from human brains (Human) were considered as 1 relative unit. Error bars represent the standard deviation of the mean values.