Table 1.
Oligonucleotide primers used for gene expression analysis.
Fig 1.
Histopathological findings in rats infected with Anisakis spp. L3.
(A) Severe submucosal hemorrhage (H) in stomach at the stomach-esophageal junction, adjacent to the site of larval penetration with two necrotic foci (asterisks) around large blood vessels (Bv) (HE, 100x); (a) inset: high-power magnification of a necrotic focus showing vascular and perivascular necrosis, surrounded by abundant neutrophil and macrophage infiltrate (arrows) (400x); (B) Stomach of a rat euthanized 24 h p.i. with abundant eosinophil infiltrate in lamina propria and around blood vessels (Bv) (arrowhead) indicating recent extravasation (HE, 100x); (b) inset: detail of abundant eosinophil infiltrate (400x); (C) Severe submucosal hemorrhage (H) in intestine with a cross-section of Anisakis spp. larva (L3) (HE, 100x); (c) inset: detail of mucosa with rare eosinophils in lamina propria (arrowheads) (HE, 400x); (D) Extensive submucosal hemorrhage (H) in caecum (HE, 100x); (d) inset: detail of mucosa with abundant eosinophil infiltration (arrowheads) in lamina propria (HE, 400x); (E) Migration of Anisakis spp. larvae (L3) through epimysial connective tissue (Epm) causing moderate to extensive hemorrhage accompanied by mixed inflammatory infiltrate, composed mostly of neutrophils and macrophages (Mf = muscle fibers) (HE, 100x); (F) Muscle tissue adjacent to site of larval migration showing mild perimysial hemorrhage (asterisks) and focal necrosis of muscle fibers (Mf) (HE, 100x); (f) inset: detail of perimysial connective tissue with two blood vessels (Bv) surrounded by moderate neutrophil infiltrate (arrow) (400x). Scale bar (A, B, C, D, E, F) = 200 μm, scale bar (inset) = 50 μm.
Fig 2.
Representative micrographs of CD3 immunolabeling in rats experimentally infected with Anisakis spp. third-stage larvae.
(A) Moderate CD3 expression on a large number of T lymphocytes (arrows) in stomach adjacent to blood vessel; (B) DAPI-stained cell nuclei; (C) Merge of (A) + (B); (D) Two lymphocytes in the intestinal submucosa showing strong CD3 expression (arrows); (E) DAPI-stained cell nuclei; (F) Merge of (D) + (E); (G) Single T lymphocyte with very strong CD3 expression in perimysial connective tissue (arrow); (H) DAPI-stained nuclei; (I) Merge of (G) + (H). Immunofluorescence, scale bar = 10 μm.
Fig 3.
Representative micrographs of iNOS immunolabeling in rats experimentally infected with Anisakis spp. third-stage larvae.
(A) Moderate to strong granular cytoplasmic expression of iNOS in a low number of cells in stomach (arrows); (B) DAPI-stained cell nuclei; (C) Merge of (A) + (B); (D) Strong iNOS expression in intestinal cells adjacent to migrating Anisakis larvae (arrows); (E) DAPI-stained cell nuclei; (F) Merge of (D) + (E); (G) Very strong iNOS expression in perimysial connective tissue in cells most likely belonging to the mononuclear phagocyte system, adjacent to blood vessel (arrows); (H) DAPI-stained nuclei; (I) Merge of (G) + (H). Immunofluorescence, scale bar = 10 μm.
Fig 4.
Representative micrographs of S100A8/A9 (MRP8+MRP14) immunolabeling in rats experimentally infected with Anisakis spp. third-stage larvae.
(A) Strong diffuse cytoplasmic labeling of S100A8/A9 in a low number of cells in the inner layer of the muscularis externa of rat stomach (arrows); (B) DAPI-stained cell nuclei; (C) Merge of (A) + (B); (D) Moderate diffuse cytoplasmic expression of S100A8/A9 in endothelial cells in intestinal submucosa; (E) DAPI-stained cell nuclei; (F) Merge of (D) + (E); (G) Very strong sarcoplasmic expression of S100A8/A9 in abdominal muscle myocytes (arrows); (H) DAPI-stained nuclei; (I) Merge of (G) + (H). Immunofluorescence, scale bar = 10 μm.
Fig 5.
Transmission electron micrographs of lesions in rats experimentally infected with Anisakis spp. third-stage larvae.
(A) Hemorrhage (H) in lamina propria of stomach with three macrophages (Mφ); (B) Rat stomach adjacent to site of larval migration through stomach wall with neutrophil (Neu) and eosinophil (Eo) infiltration; (C) Rat intestine adjacent to site of larval migration through intestinal wall with a single erythrocyte (Er) indicative of hemorrhage and eosinophil (Eo) and macrophage (Mφ) infiltration; bottom of picture shows elaborate endoplasmic reticulum indicative of a plasma cell (Pc). Inset: numerous elongated fungus-like organisms with electron lucent intracytoplasmic granules; (D) Mixed inflammatory infiltrate of rat caecum composed of neutrophils (Neu), eosinophils (Eo) and macrophages (Mφ) interspersed between collagen fibers (Cf) and mixed with non-nucleated erythrocytes (Er) indicating hemorrhage; (E) Necrotic myocytes of abdominal muscle with fragmented muscle fibers (Mf); few neutrophils with outspread pseudopodia migrating over muscle fibers (Mf) and a single macrophage (Mφ) are visible; (F) Mixed inflammatory infiltrate of rat muscle dominated by neutrophils (Neu), and few eosinophils (Eo), macrophages (Mφ), and erythrocytes (Er), indicating hemorrhage. Osmium tetroxide/lead citrate, scale bar: (A, E, F) = 10 μm, scale bar (B, C) = 2 μm, scale bar (D, C inset) = 5 μm.
Fig 6.
Expression of target genes in tissues of rats experimentally infected with Anisakis spp. third-stage larvae.
(A) Distribution of target gene expression in stomach, intestine, and visceral adipose tissues grouped according to state (infected or uninfected). Box plots represent distribution of log2-normalized expression; (B) Log2-transformed fold changes of target gene expression with respective adjusted p-values.
Fig 7.
miRNAs expression in stomach and intestine of rats experimentally infected with Anisakis spp. third-stage larvae.
(A) Two-dimensional hierarchical clustering and heatmap of log2-normalized miRNA expression (Manhattan distance), grouped according to their expression profiles, tissue of origin (stomach and intestine), and tissue state (infected or uninfected). Color key ranges from white to light grey for downregulated (−2 to 0, Z-scores) and from light grey to black for upregulated (0–2, Z-scores) miRNAs. Only differentially expressed miRNAs (p < 0.05) in stomach and intestine are listed on the right (adjusted p-value < 0.05 in bold). (RN7_3, RN8_1, RN9_6: stomach, infected; RN7_3K, RN8_1K, RN9_6K: stomach, uninfected; RN6_4, RN7_5, RN10_3: intestine, infected; RN6_4K, RN7_5K: intestine, uninfected); (B) Log2-transformed fold changes with respective p-values and adjusted p-values of differentially expressed miRNAs in stomach and intestine.