Fig 1.
Representative histological images of the liver from uninfected (A) and naturally S. mansoni-infected N. squamipes (B). A normal hepatic tissue is seen in (A) while typical granulomas at different developmental stages (circles) are observed in (B). One mature granuloma characterized by a central parasite egg surrounded by a dense population of inflammatory cells is shown in high magnification in (Bi). Eosinophils with characteristic acidophilic cytoplasm and mononuclear cells are clearly observed. In (C), the layer formed by collagen fibers is seen at the outer zone of the granuloma. Liver fragments (n = 3 animals from each group) were fixed in buffered paraformaldehyde, embedded in glycolmethacrylate resin and cut into 3 μm-thick sections, which were stained with hematoxylin-eosin (A, B) or Gomori’s trichrome (C). Scale bar, 60 μm (A, B); 120 μm (Bi); 100 μm (C).
Fig 2.
Excessive LD accumulation occurs in the liver of naturally S. mansoni-infected N. squamipes.
In (A and C), the hepatic tissue shows putative LDs, negatively stained with alcoholic hematoxylin-eosin. (B and D), ORO staining confirms the presence of numerous LDs seen as round organelles (stained in red) and distributed within hepatocytes. In (Di), a hepatocyte is seen in high magnification. (E, F) Quantitative analyses of LD numbers in the hepatic tissue. ORO staining was performed on cryosections from liver fragments fixed in buffered paraformaldehyde. LD quantifications were done in a slide scanner using Pannoramic Viewer and Histoquant softwares. A total of 1,500,000 μm2 of tissue area was evaluated per animal, with a total of 4,500,000 μm2 of tissue area analyzed per group (n = 3 animals). Data represent mean ± S.E.M. ****P < 0.0001, ** P < 0.004 versus uninfected group. Scale bar, 40 μm (A,D); 50 μm (B); 75 μm (C); 8 μm (Di).
Fig 3.
Ultrastructure of LDs formed in the liver of uninfected and naturally S. mansoni-infected N. squamipes.
(A-C) Electron micrographs of the hepatic tissue reveal electron lucent LDs with varied sizes within hepatocytes in both control (A) and infected (B, C) livers. Note in (B), a giant LD (arrow) in the hepatocyte cytoplasm. (D) TEM quantitative analyses show a high number of LDs per tissue area (P < 0.002). The range of LD diameter is shown in (E). Scale bar, 9 μm (A-C). Liver fragments were fixed in a mixture of paraformaldehyde/glutaraldehyde and processed for TEM. Quantitative analyses were performed in a total of 34,000 μm2 of hepatic tissue (17,000 μm2 from uninfected and 17,000 μm2 from infected animals) using the software ImageJ. Data represent mean ± S.E.M. ***P < 0.0001 for infected versus uninfected group.
Fig 4.
Serum lipid profile of uninfected and naturally S. mansoni-infected N. squamipes.
(A) The levels of total cholesterol and triglycerides are significantly increased in the infected group in comparison to the uninfected animals (****P < 0.0001, n = 13 animals per group). (B) Serum lipids increase proportionally to the increase of LD numbers in the liver. LDs were counted in the liver after ORO staining as shown in Fig 2. Data represent minimum-median-maximum (A) and mean ± S.D (B).
Fig 5.
Raman spectra reveal high content of polyunsaturated fatty acids (PUFAs) in both uninfected and infected liver of N. squamipes.
(A) The band at 3015 cm-1 is characteristic of PUFAs and is more prominent in arachidonic acid (AA) when compared to other PUFAs. The degree of unsaturation is shown in (B). Liver fragments of N. squamipes naturally infected and uninfected were fixed and analyzed by Raman spectroscopy without labeling. AA spectrum was obtained from pure AA (catalog number A3555, Sigma-Aldrich) diluted in ethanol. Data are representative of 3 independent experiments.
Fig 6.
MALDI-TOF mass spectra of liver tissues reveal high concentration of arachidonic acid (AA) in naturally S. mansoni-infected N. squamipes.
(A, B) Mass spectra, from 301 to 307 m/z range showing peaks attributed to sodium adduct of the linoleic acid [M + Na]+ (m/z 303.03), AA (m/z 304.24) and sodium adduct of the oleic acid [M + Na]+ (m/z 305.26). (C, D) Mass spectra from 324 to 330 m/z range. The peak observed at 327 m/z represents sodium adduct of the AA [M + Na]+. Liver sections from uninfected and naturally S. mansoni-infected N. squamipes (n = 3 sections from each group) were cut on a cryostat (50 μm thickness) and analyzed without any labeling.
Table 1.
Serum transaminases of uninfected and S. mansoni-infected N. squamipes.