Analysis of AA amyloid protein in fecal matter from cheetahs with amyloidosis. (a) AA amyloid fibril fractions were analyzed by Western blot. Lane 1, sample (20 μg) from liver of C68. Lane 2, sample (20 μg) from feces of C78. Lane 3, 100 μg of protein from lyophilized whole urine of C68. Lane 4, pellets centrifuged at 100,000 × g from 4.0 ml of urine of C68. (b) Amino acid sequences of AA amyloid proteins from the liver and the feces corresponding to monomer detected in Fig. 2a. The fecal AA amyloid protein lacked several amino acids from both the N and C termini compared with the liver AA amyloid protein. (c) The AA amyloid fibrils from the liver (A) and the feces (B) were observed by transmission electron microscopy. Open triangles indicate characteristic amyloid fibrils; filled triangles represent fecal-specific fibrils. (Scale bar, 100 nm.)

Quantitation of transmissibility of AA amyloid fibrils from feces. (a) The degree of amyloid deposition in spleens of mice induced by fecal amyloid fibril fractions (filled squares) (1 μg, 10 μg, and 100 μg) or the liver fractions (open squares) (10 μg) was determined in Congo red-stained tissue sections (four mice per group). (b) The degree of AA deposition in induced mice was determined by isolation of AA amyloid fibril fractions from the spleens of mice in each group (filled squares, fecal; open diamonds, liver) followed by Western blot analysis (20 μg of protein per well) and quantification using National Institutes of Health Images. The means and SE were determined by the relative ratios of AA amyloid protein levels in each group versus the group receiving 10 μg of amyloid fibrils fraction from the feces (*, P < 0.05; **, P < 0.01).

Cheetah AA amyloid fibril proteins and specific antiserum. (a) The isolated AA amyloid fibril fractions from the livers of C68 and C66 (20 μg) were applied to 16.5% Tris·tricine SDS/PAGE gel followed by CBB staining. M, molecular weight markers. Molecular masses of two main and two faint bands corresponded to the AA monomer. (b) Amino acid sequencing from four bands in C68 sample. AA amyloid fragments with different lengths were obtained. The amyloid fibril fraction from C68 was used as antigen to produce specific anti-cheetah AA antiserum. (c) Amyloid deposition in the liver of C68 was detected in Congo red-stained sections by standard microscopy (Left), by green birefringence under polarized microscopy (Center), and by immunohistochemical staining (Right). The anti-cheetah AA antiserum was used as primary antibody (dilution 1:1,000). (Magnification, ×200.) (d) AA amyloid fibril fractions from the liver of C68 (lane 1), human (lane 2), mouse (lane 3), cow (lane 4), and murine AApoAII(C) amyloid fibril fraction (lane 5) were used (12 μg of protein per well) to perform Western blot analysis with anti-cheetah AA antiserum as primary antibody (dilution 1:3,000).

High transmissibility of feces from the cheetah with amyloidosis. (a) Fecal (C68 and C67) and liver (C68) AA amyloid fibril fractions were untreated (N), or treated by guanidine-hydrochloride (G) or formic acid (F) and injected into mice to induce AA amyloidosis. Equal quantities of amyloid fractions (100 μg) were used in each experiment. The degree of amyloidosis was determined by the amyloid deposition observed in Congo red-stained sections of the spleen (*, P < 0.05; **, P < 0.01). (b) AA deposition in the spleens of mice exposed to untreated fecal fibrils (A and B) (grade 4) or untreated liver fibrils (G and H) (grade 3). Deposition observed in mice exposed to guanidine-hydrochloride treated fecal fibrils (C and D) (grade 1) or liver fibrils (I and J) (grade 3) and to formic acid treated fecal fibrils (E and F) (grade 0) or liver fibrils (K and L) (grade 1). Deposition was detected by green birefringence in Congo red-stained sections under polarized microscopy (Upper) and immunohistochemical staining with anti-mouse AA antiserum (Lower). (Magnification, × 200.)

AA amyloid protein in the fecal amyloid fibril fraction is required for transmission. (a) The fecal amyloid fibril fraction from C90 was untreated (UT), immunodepleted with normal rabbit IgG (ID-IgG) or anti-cheetah AA antiserum (ID-AA) for one cycle (Left) or five cycles (Right) and then were separated with 16.5% SDS/PAGE followed by immunoblotting with anti-cheetah AA antiserum (10 μl per well). (b) UT and ID-IgG or ID-AA for five cycles of immunodepletion were used to induce AA amyloidosis in C57BL/6 mice (four mice per group). The amount of amyloid deposited was graded in Congo red-stained sections. (c) AA amyloid protein levels in the spleens from mice exposed to UT, ID-IgG, or ID-AA for five cycles of immunodepletion. AA amyloid proteins were isolated from each group mice, and Western blots were analyzed (20 μg of protein per well) followed by quantitative analysis using National Institutes of Health Image. The means and SE were determined by the relative ratios of AA amyloid protein levels for each induced group versus the group induced by the untreated sample (*, P < 0.05; **, P < 0.01).