Fig 1.
SEM of the cecum with Trichuris muris disrupting the intestinal mucosa after 10 and 17 days of infection and bacterial adhesion.
(A) SEM image of the cecum of mice infected with Trichuris muris 10 days after infection. (B, C and D) SEM image of the cecum of a mouse infected with Trichuris muris for 17 days. (A) SEM image of the intestinal mucosa and the route taken by the parasite (asterisk) that subsequently penetrates into the mucosa and breaks parts of the intestinal mucosa, with bacteria close to the rupture site (dotted square). The inset shows the parasite penetrating the tissue at higher magnification and carrying bacteria (arrow) present on the mucosal surface (Mu), revealing the path taken by the parasite in the intestinal mucosa. The scale bars represent 10 µm; (inset) 5 µm. (B) Cecum fracture showing the intestinal mucosa and the parasite (asterisk) inserted, forming the syncytial tunnel, goblet cells (Gc), rupturing the intestinal mucosa, and carrying bacteria (arrows) in the mucosa. The scale bar represents 20 µm. The inset shows the parasite (asterisk) penetrating the mucosa and carrying bacteria (arrows) into the mucosa at higher magnification. Scale bar represents 5 µm. (C) Parasite (asterisk) inserted in the intestinal mucosa with bacteria adhered (arrow) to its body and to the surface of the intestinal mucosa. The scale bar represents 20 µm. (D) Cecum fracture showing the nematode (asterisk) and mucosa (Mu) and submucosa (Sm) layers with bacteria in the submucosa (arrows). The scale bar represents 20 µm. Insets of the invasive bacteria (arrows) inside the submucosa (Sm). The scale bar represents 5 µm.
Fig 2.
SEM of the cecum with Trichuris muris disrupting the intestinal mucosa after 17 and 22 days of infection and bacteria invading the submucosa.
(A and B) SEM image of the cecum of a mice infected with Trichuris muris for 17 days. (C, D, E and F) SEM image of the cecum of a mouse infected with Trichuris muris for 22 days. (A) Parasite (asterisk) penetrating into the intestinal mucosa, causing ruptures (arrowhead), breaking several regions of tissue during migration throughout the syncytial tunnel. Scale bar represents 30 µm. (B) Anterior end of the nematode showing the stylet (s) and bacteria on the mucosa surface (arrows). Scale bar represents 10 µm. (C) Fractured cecum showing the three layers of the mucosa (Mu), submucosa (Sm) and muscular layer (M), and the parasite (asterisk) penetrating into the mucosa, forming the syncytial tunnel (tu) and causing ruptures (arrowhead) on the tissue surface and high bacterial levels in the submucosa. The scale bar represents 100 µm. (D) Details of the parasite (asterisk) inside the tunnel (tu) in a cross-section of mucosa (Mu). Scale bar represents 10 µm. (E) Details of the bacteria inside the intestinal submucosa (Sm). Scale bar represents 10 µm. (F) Detail of the parasite (asterisk) breaking the intestinal mucosa (Mu); the arrowheads indicate bacteria adhering to the nematode cuticle and on the microvilli of the intestinal mucosa (arrows). Scale bar represents 10 µm.
Fig 3.
Total number of peritoneal cells.
Variation in the total number of peritoneal cells observed in the Trichuris muris infection group. The significance of differences between the noninfected and infected groups (n = 5) was determined via Student’s t test, and asterisks indicate statistically significant differences. * p value ≤ 0.05.
Fig 4.
Histology of the cecum after 90 minutes and 10 and 17 days of infection by Trichuris muris.
Histological section of the large intestine (cecum) stained with hematoxylin and eosin showing the morphology and morphometry of the large intestine layers. (A, C, E) Group not infected and (B, D, F) infected with Trichuris muris. (A, B) 90 minutes after infection. (C, D) 10 days after infection. (E, F) 17 days after infection. An increase in the thickness of the mucosa (Mu), submucosa (Sm) and muscle layers (M) and increased infiltration of inflammation in the submucosa were observed beginning on the 10th day of infection. Scale bar represents 100 µm.
Fig 5.
Histology of the cecum after 22 and 35 days of infection by T. muris.
Histological section of the large intestine (cecum) stained with hematoxylin and eosin showing the morphology and morphometry of the large intestine layers. (A, C) Noninfected group; (B, D) infected with Trichuris muris. (A, B) After 22 days of infection. (C, D) After 35 days of infection. (E) Morphometric analyses of the mucosa (Mu); (F) morphometric analyses of the submucosa (Sm); (G) morphometric analyses of the muscularis layers (M), showing an increase from the 10th day of infection to the 35th day. The significance of differences between the noninfected and infected groups (n = 10) was determined via Student’s t test, and asterisks indicate statistically significant differences. * p value ≤ 0.05. Scale bar represents 100 µm.
Table 1.
Intestinal mucosa morphological analysis.
Table 2.
Intestinal submucosa morphological analysis.
Table 3.
Quantification of goblet cells and enterocytes.
Fig 6.
Increased number of goblet cells and enterocytes from the 17th day of infection.
Histological sections of the large intestine (cecum) of 17-day-infected mice stained with PAS. (A) Noninfected mice showing mucosa (Mu) with few goblet cells and enterocytes (cells color pink), thin submucosa (Sm) and a muscle layer (M). (B) Mice infected with Trichuris muris presented an increase in the number of goblet cells and enterocytes (pink) in the mucosa (Mu), submucosa (Sm) and muscular layer (M). Scale bar represents 50 µm.
Fig 7.
Quantification of the amount of feces.
Graph illustrating the percentage of feces retained in the cecum of the mice. Between the 22nd and 35th days of infection, fewer feces were retained in the cecum of the mice infected with Trichuris muris. The significance of differences between the noninfected and infected groups (n = 5) was determined via Student’s t test, and asterisks indicate statistically significant differences. * p value ≤ 0.05.
Fig 8.
Th1 profile of cytokines present in the cecum and mesenteric lymph nodes throughout infection.
Cytokine Th1 secretion in the cecum and mesenteric lymph nodes of noninfected and infected mice by Trichuris muris. The results are expressed in picograms per milliliter. After isolation and homogenization, the proteins were measured via an ELISA kit. The significance of differences between the noninfected and infected groups (n = 4) was determined via Student’s t test, and asterisks indicate statistically significant differences. * p value ≤ 0.05.
Fig 9.
Th2 profile of cytokines present in the cecum and mesenteric lymph nodes throughout infection.
Cytokine Th2 secretion in the cecum and mesenteric lymph nodes of noninfected and infected mice by Trichuris muris. The results are expressed in picograms per milliliter. After isolation and homogenization, the cytokines were measured via an ELISA kit. The significance of differences between the noninfected and infected groups (n = 4) was determined via Student’s t test, and asterisks indicate statistically significant differences. * p value ≤ 0.05.
Fig 10.
Cytokine secretion in peritoneal macrophages.
Cytokine secretion in peritoneal macrophages of noninfected and infected mice by Trichuris muris. After isolation and homogenization, the cytokines were measured via an ELISA kit. The significance of differences between the noninfected and infected groups (n = 4) was determined via Student’s t test, and asterisks indicate statistically significant differences. * p value ≤ 0.05.
Fig 11.
Nitric oxide dosage of peritoneal macrophages.
Cultures of peritoneal macrophages from noninfected and infected Trichuris muris-infected mice were maintained under culture conditions for 24h and stimulated with LPS (3 g/ml), and NO-2 was measured. The significance of differences between the noninfected and infected groups (n = 4) was determined via Student’s t test, and asterisks indicate statistically significant differences. * p value ≤ 0.05.
Table 4.
Spleen data obtained throughout the infection.
Fig 12.
Spleen histology showing megakaryocytes.
Histological section of the spleen, which was stained with hematoxylin and eosin from a 35-day-old mouse infected with Trichuris muris, showing megakaryocytes (arrow). Scale bar represents 20 μm.
Fig 13.
Fluorescence in situ hybridization (FISH) of sections from noninfected and Trichuris muris-infected mice revealed bacterial invasion beginning on the 17th day of infection.
Merged images showing bacteria (green) and host tissue cells (DAPI). (A) Noninfected mouse cecum after 35 days, showing three tissue layers, the mucosa (Mu), submucosa (Sm) and muscle layers (M), with bacteria found only in the intestinal mucosa layer. Scale bar represents 20 µm. (B) Mouse cecum after 17 days of infection, showing the intestinal mucosa (Mu) and submucosa (Sm) layers and bacterial invasion in the submucosa. Scale bar represents 20 µm. (C) The inset of (B) shows the mucosa (Mu) and submucosa (Sm), with bacteria (arrow) in the submucosa. Scale bar represents 10 µm. (D) The inset of (C) is shown at higher magnification, with the submucosa with coccus and bacillus bacteria (arrow) invading the tissue, as indicated by the arrow. Scale bar represents 5 µm. (E) Mouse cecum after 22 days of infection showing the parasite (asterisk) inserted into the intestinal mucosa (Mu), bacteria (arrow) close to the parasite and invading the intestinal submucosa (Sm), and the muscle layer is large. Scale bar represents 10 µm.
Fig 14.
Images of the 3D model obtained via light sheet microscopy showing the infected intestine in the chronic phase of infection.
(A–D) Series of the infected intestine model with transparency performed via Zen software, showing different angles of the mucosa (Mu) and nematode (asterisk). Scale bar represents 10 µm. (E, F) Three-dimensional model performed via Amira’s automatic segmentation isosurface showing the nematode (asterisk) and mucosa surface (Mu). Scale bar represents 300 µm.
Fig 15.
Three-dimensional modeling and color scanning electron microscopy (SEM) results were integrated to explain the bacterial translocation process.
(A) Micro-CT image of the cecum ofa 35-day-old mouse showing epithelial tunnels (tu) in different parts of the surface mucosa (Mu) and the posterior region of the parasite in the lumen (asterisk). Scale bar represents 500 µm. (B) Three-dimensional model based on Amira’s automatic segmentation isosurface showing the penetrating nematode (tu), tissue breaks (arrowhead), and posterior region of the parasite (asterisk). Inset lateral view of the model showing the three intestinal layers, including the mucosa (Mu), submucosa (Sm), muscular layer (M), and the nematode (asterisk). The scale bar represents 1 mm. Inserted (B) images showing the mucosa (Mu), submucosa (Sm) and muscle layer (M) at lower magnification and parasites in the lumen. The scale bar represents 1 mm. (C) SEM image of the mucosa surface (pink color) showing the anterior region of the nematode (yellow color), tissue breaks on the mucosa (arrowhead) exposing the nematode cuticle, nematode egg on the surface (e), and bacteria (blue color – dotted circles). The scale bar represents 100 µm. (D, E) Rendered three-dimensional models integrating results and showing the nematode (yellow color), tissue breaks (arrowheads), epithelial tunnels (tu), and bacteria (green and purple - dotted circles) on the mucosal surface (pink) and on the nematode body. Scale bars represent 500 µm.
Fig 16.
Dynamics of infection development.
Scheme showing the intricate interplay among host immune responses, the timing of bacterial translocation in the gut, changes in the intestinal layers, spleen architecture, peritoneal macrophages, and parasite survival strategies during the development of Trichuris muris infection. Created with BioRender.com and then modified.