Subclinical bovine spongiform encephalopathy infection in transgenic mice expressing porcine prion protein

Abstract

The bovine-porcine species barrier to bovine spongiform encephalopathy (BSE) infection was explored by generating transgenic mouse lines expressing the porcine prion protein (PrP) gene. All of the porcine transgenic (poTg) mice showed clinical signs of BSE after intracerebral inoculation with a high-titer BSE inoculum. The protease-resistant PrP (PrP(res)) was detected in 14% (3 of 22) of the BSE-infected poTg mice by immunohistochemical or immunoblot analysis. Despite being able to infect 42% (5 of 12) of control mice, a low-dose BSE inoculum failed to penetrate the species barrier in our poTg mouse model. The findings of these infectivity studies suggest that there is a strong species barrier between cows and pigs. However, after second-passage infection of poTg mice using brain homogenates of BSE-inoculated mice scoring negative for the incoming prion protein as inoculum, it was possible to detect the presence of the infectious agent. Thus, porcine-adapted BSE inocula were efficient at infecting poTg mice, giving rise to an incubation period substantially reduced from 300 to 177 d after inoculation and to the presence of PrP(res) in 100% (21 of 21) of the mice. We were therefore able to conclude that initial exposure to the bovine prion may lead to subclinical infection such that brain homogenates from poTg mice classified as uninfected on the basis of the absence of PrP(res) are infectious when used to reinoculate poTg mice. Collectively, our findings suggest that these poTg mice could be used as a sensitive bioassay model for prion detection in pigs.

Figure 1.

Expression of the porcine PrP protein in heterozygous (mo^+/-po^+/-) poTg lines. Immunoblotting of transgenic mouse brain extracts from Tg lines 001, 011, 016, 027, 040, 046, and 101 using the monoclonal antibodies 2A11 (A) or FH11 (B). Serial dilution of homozygous (mo^-/-po^+/+) poTg mouse lines (001 and 027) and porcine brain homogenates were analyzed by Western blotting using monoclonal antibody 2A11 (C). Po, Porcine brain extract; Mo, B6CBA × 129/Ola mouse brain extract. Equivalent amounts of protein were loaded onto each lane. Relative molecular mass is given in kilodaltons. MW, Molecular weight.

Figure 2.

Detection of the PrP^res protein in inoculated poTg mice. A, Western blot of poTg027 brain extracts from mice inoculated intracerebrally with the different inocula. The PrP^res protein was only detected in one of five boTgBSE₁-inoculated mice. B, Western blot of poTg001 and poTg027 brain extracts of mice inoculated intracerebrally with the poTgBSE₁ inoculum. All of the mice inoculated showed PrP^res. C, Western blot of boTgBSE₁, poTgBSE₁ passage 1, and poTgBSE₁ passage 2 brain homogenates digested with PK. Monoclonal antibody 2A11 was used at a 1:2000 dilution. Relative molecular mass is given in kilodaltons. MW, Molecular weight; pK, proteinase K.

Figure 4.

Neuropathological appearance of brain sections of PBS-inoculated poTg001 mice (540 dpi; A), BSE₁-inoculated poTg001 mice (540 dpi; B), and BSE₂-inoculated poTg001 mice (488 dpi; C). The numbers 1-3 correspond to sections of the thalamus, and 4-6 correspond to cerebellar sections. 3, 6, Hematoxylineosin staining; 2, 5, anti-GFAP immunostaining; 1, 4, 2A11 mAb immunostaining. Note the fine granular immunolabeling in the neuropil and glial pattern in the thalamus (C₁) and granular immunolabeling in neuronal bodies of the cerebellar nuclei (C₄). Scale bars, 50 μm.

Figure 3.

Quantification of the PrP^res content of the different inocula used. Serial dilution of 1% brain homogenates was Western blotted using mAb 2A11. BSE₁, A pool of brainstem material from 49 BSE-infected cattle (TSE/08/59) supplied by the VLA; boTgBSE₁, a pool of material from bovine transgenic mice (line 110) infected with first-passage BSE₁ inoculum generated in our laboratory; BSE₂, brainstem material from one BSE-infected cow supplied by the VLA. Monoclonal antibody 2A11 was used at a 1:2000 dilution. Relative molecular mass is given in kilodaltons. MW, Molecular weight.

Figure 5.

Neuropathological appearance of brain sections of PBS-inoculated poTg001 mice (180 dpi; A), poTgBSE₁-inoculated poTg001 mice (180 dpi; B), and BSE₁-inoculated boTg110 mice (250 dpi; C). The numbers 1-3 correspond to sections of the thalamus, and 4-6 correspond to cerebellar sections. 3, 6, Hematoxylineosin staining; 2, 5, anti-GFAP immunostaining; 1, 4, 2A11 mAb immunostaining. Note the fine granular immunolabeling in the neuropil and glial pattern in the thalamus (B₁) and granular immunolabeling in neuronal bodies of the cerebellar nuclei (B₄). Scale bars, 50 μm.

Figure 6.

Alignments of amino acid sequences in bovine, porcine, and murine PrPs. White letters with gray shading represent amino acid changes with respect to the bovine sequence. Vertical boxes indicate the protein X epitope.