Gut-Associated Lymphoid Tissue: A Key Tissue Inside the Mucosal Immune System of Hens Immunized with Escherichia coli F4

Abstract

Immunoglobulin Y (IgY) is the predominant antibody found in hen's (Gallus domesticus) egg yolk. This antibody, developed against several microorganisms in hen egg yolk, has been successfully used as an alternative to immunoglobulins from mammals for use in immunodiagnostics and immunotherapy. Enteropathogenic Escherichia coli (E.coli) F₄ is the main etiological agent associated with swine neonatal diarrhea, and it causes notable economic losses in swine production. The aim of the present study was to evaluate the relationship between humoral immune response and the activation of gut-associated lymphoid tissue (GALT) in laying hens intramuscularly immunized with E. coli F₄. Adult laying Shaver hens were immunized with a bacterin based on an inactivated lysate E. coli F₄ strain that was originally isolated from neonatal piglet diarrhea, following a recommended schedule. The percentage of B lymphocytes in blood and spleen homogenates was determined by flow cytometry. Villi histomorphometry and the size of germinal centers (GC) activated in GALT and the spleen were measured in histological samples either stained with hematoxylin/eosin or through immunofluorescence. Antibody and isotype-specific antibodies in serum and egg yolk were measured using indirect enzyme-linked immunosorbent assay (ELISA). Secretory and serum immunoglobulin A (IgA) were measured by ELISA tests. Laying hen with intramuscular immunization with E. coli F₄ lysate, activated both mucosal and systemic protection. Mucosal protection was provided through B lymphocytes, and most of them were activated on Peyer's patches and esophageal tonsils, in GALT. Furthermore, increased B lymphocyte number in the lamina propria of the gut, and increased intraepithelial plasmatic cell number, produced high levels of mucosal IgA. Activated B lymphocytes interacted with absorptive cells, immune cells, and microbiota in the gut, producing signals that were translated into a powerful physical defense by producing a greater volume of mucin from an increased number of goblet cells. Systemic protection was provided through B lymphocyte activation of spleen GC, which produced hugely specific IgY serum levels. One week later, this specific IgY was deposited in the yolk. This suggests that GALT is a key immunologic tissue inside the mucosal immune system, acting as the "command center" for humoral reaction.

Keywords: B lymphocytes; Escherichia coli F4; flow cytometry; gut-associated lymphatic tissue; hen; histomorphometry; immunoglobulin A; immunoglobulin Y technology.

Figure 1

Peyer’s patches microphotography, with marked (abundant) lymphocyte production in intestinal histological sections in the F4 group and control group. (A) Immunofluorescence image, stained with the B lymphocyte surface antigen AV20-FitC control group. Magnification: 400×. (B) Immunofluorescence image, stained with the B lymphocyte surface antigen AV20-FitC. F₄ group. Into circle highlight a completely activated germinal center. Magnification: 400× (C) Peyer’s patches of control group hens, stained with hematoxylin-eosin. Magnification: 100×. (D) Peyer’s patches of F₄ group hens, stained with hematoxylin-eosin. Magnification: 100×.

Figure 2

Intestinal villi. Intestinal histopathology microphotography in the control group (A) and F₄ group (B) of laying hens. Into F₄ group the villi with abundant goblet cells are highlighted (a) next to the mucus layer with an increased number of lymphocytes inside the villi (b) stained with hematoxylin/eosin. Magnification: 400×.

Figure 3

Immunoglobulin A (IgA) concentration levels. IgA concentration levels in laying hens from 23 to 42 weeks in the F₄ (F₄) and Control (C) groups. ***P < 0.001. Arrows indicate immunizations.

Figure 4

B lymphocyte number in blood. B lymphocyte percentages in laying hens in Control and F₄ groups at the end of the assay (43 weeks old). *P < 0.05.

Figure 5

B lymphocyte number in the spleen. B lymphocyte percentages in laying hens in Control and F₄ groups at the end of the assay (43 weeks old). *P < 0.05.

Figure 6

Total immunoglobulin levels. Serum and yolk levels of total immunoglobulin Y (IgY) in both the F₄ and Control groups from 23 to 42 weeks *P < 0.05, ***P < 0.001. Arrows indicate immunizations

Figure 7

Anti-Escherichia coli F4-immunoglobulin levels. Serum and yolk levels of anti-E. coli F₄-IgY in the F₄ and Control groups from 23 to 42 weeks *P < 0.05, ***P < 0.001. Arrows indicate immunizations.